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Beyond icosahedral symmetry in packings of proteins in spherical shells.

Majid Mosayebi, Deborah K. Shoemark, Jordan M. Fletcher, Richard B. Sessions, Noah Linden, Derek N. Woolfson and Tanniemola B. Liverpool

PNAS 2017 vol 114 pp 9014-9019 DOI: 10.1073/pnas.1706825114

The formation of quasi-spherical cages from protein building blocks is a remarkable self-assembly process in many natural systems, where a small number of elementary building blocks are assembled to build a highly symmetric icosahedral cage. In turn, this has inspired synthetic biologists to design de novo protein cages. We use simple models, on multiple scales, to investigate the self-assembly of a spherical cage, focusing on the regularity of the packing of protein-like objects on the surface. Using building blocks, which are able to pack with icosahedral symmetry, we examine how stable these highly symmetric structures are to perturbations that may arise from the interplay between flexibility of the interacting blocks and entropic effects. We find that, in the presence of those perturbations, icosahedral packing is not the most stable arrangement for a wide range of parameters; rather disordered structures are found to be the most stable. Our results suggest that (i) many designed, or even natural, protein cages may not be regular in the presence of those perturbations and (ii) optimizing those flexibilities can be a possible design strategy to obtain regular synthetic cages with full control over their surface properties.


CCBuilder 2.0: Powerful and accessible coiled-coil modelling

Christopher W. Wood and Derek N. Woolfson

Protein Science 2017 DOI: 10.1002/pro.3279

The increased availability of user-friendly and accessible computational tools for biomo-lecular modeling would expand the reach and application of biomolecular engineering and design.For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The alpha-helical coiled-coil provides one such example, which represents 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of alpha-helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both alpha-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "darkmatter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely.


Miniprotein Design: Past, Present, and Prospects

Emily G. Baker, Gail J. Bartlett, Kathryn L. Porter Goff and Derek N. Woolfson

Accounts of Chemical Research 2017 v 50 pp 2085-2092. DOI: 10.1021/acs.accounts.7b00186

The design and study of miniproteins, that is, polypeptide chains <40 amino acids in length that adopt defined and stable 3D structures, is resurgent. Miniprotein offer possibilities for reducing the complexity of larger proteins and so present new routes to studying sequence-to-structure and sequence-to-stability relationships in proteins generally. They also provide modules for protein design by pieces and, with this, prospects for building more-complex or even entirely new protein structures. In addition, miniproteins are useful scaffolds for templating functional domains, for example, those involved in protein−protein interactions, catalysis, and biomolecular binding, leading to potential applications in biotechnology and medicine. Here we select examples from almost four decades of miniprotein design, development, and dissection. Simply because of the word limit for this Account, we focus on miniproteins that are cooperatively folded monomers in solution and not stabilized by cross-linking or metal binding. In these cases, the optimization of noncovalent interactions is even more critical for the maintenance of the folded states than in larger proteins. Our chronology and catalogue highlights themes in miniproteins, which we explore further and begin to put on a firmer footing through an analysis of the miniprotein structures that have been deposited in the Protein Data Bank (PDB) thus far. Specifically, and compared with larger proteins, miniproteins generally have a lower proportion of residues in regular secondary structure elements (alpha helices, beta strands, and polyproline-II helices) and, concomitantly, more residues in well-structured loops. This allows distortions of the backbone enabling mini-hydrophobic cores to be made. This also contrasts with larger proteins, which can achieve hydrophobic cores through tertiary contacts between distant regions of sequence. On average, miniproteins have a higher proportion of aromatic residues than larger proteins, and specifically electron-rich Trp and Tyr, which are often found in combination with Pro and Arg to render networks of CH-pi or cation-pi interactions. Miniproteins also have a higher proportion of the long-chain charged amino acids (Arg, Glu, and Lys), which presumably reflects salt-bridge formation and their greater surface area-to-volume ratio. Together, these amino-acid preferences appear to support greater densities of noncovalent interactions in miniproteins compared with larger proteins. We anticipate that with recent developments such as parametric protein design, it will become increasingly routine to use computation to generate and evaluate models for miniproteins in silico ahead of experimental studies. This could include accessing new structures comprising secondary structure elements linked in previously unseen configurations. The improved understanding of the noncovalent interactions that stabilize the folded states of such miniproteins that we are witnessing through both in-depth bioinformatics analyses and experimental testing will feed these computational protein designs. With this in mind, we can expect a new and exciting era for miniprotein design, study, and application.


How do miniproteins fold?

Derek N. Woolfson, Emily G. Baker and Gail J. Bartlett

Science 2017 DOI: 10.1126/science.aan6864

Perspective on Rocklin et. al. "Global analysis of protein folding using massively parallel design, synthesis, and testing.", DOI: 10.1126/science.aan0693

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Decorating Self-Assembled Peptide Cages with Proteins

James F. Ross, Angela Bridges, Jordan M. Fletcher, Deborah Shoemark, Dominic Alibhai, Harriet E. V. Bray, Joseph L. Beesley, William M. Dawson, Lorna R. Hodgson, Judith Mantell, Paul Verkade, Colin M. Edge, Richard B. Sessions, David Tew, and Derek N. Woolfson

ACS Nano 2017 DOI: 10.1021/acsnano.7b02368

An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide–protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.


Construction of a Chassis for a Tripartite Protein-Based Molecular Motor

Lara S. R. Small, Marc Bruning, Andrew R. Thomson, Aimee L. Boyle, Roberta B. Davies, Paul M. G. Curmi, Nancy R. Forde, Heiner Linke, Derek N. Woolfson, and Elizabeth H. C. Bromley

ACS Synthetic Biology 2017 DOI: 10.1021/acssynbio.7b00037

Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultra-centrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.


Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Marcin Wolny, Matthew Batchelor, Gail J. Bartlett, Emily G. Baker, Marta Kurzawa, Peter J. Knight, Lorna Dougan, Derek N. Woolfson, Emanuele Paci, Michelle Peckham

Scientific Reports 2017 DOI: 10.1038/srep44341

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.


Membrane-spanning alpha-helical barrels as tractable protein-design targets

Ai Niitsu, Jack W. Heal, Kerstin Fauland, Andrew R. Thomson, Derek N. Woolfson

Philosophical Transactions of the Royal Society B 2017 doi: 10.1098/rstb.2016.0213

The rational (de novo) design of membrane-spanning proteins lags behind that for water-soluble globular proteins. This is due to gaps in our knowledge of membrane-protein structure, and experimental difficulties in studying such proteins compared to water-soluble counterparts. One limiting factor is the small number of experimentally determined three-dimensional structures for transmembrane proteins. By contrast, many tens of thousands of globular protein structures provide a rich source of ‘scaffolds’ for protein design, and the means to garner sequence-to-structure relationships to guide the design process. The α-helical coiled coil is a protein-structure element found in both globular and membrane proteins, where it cements a variety of helix–helix interactions and helical bundles. Our deep understanding of coiled coils has enabled a large number of successful de novo designs. For one class, the α-helical barrels—that is, symmetric bundles of five or more helices with central accessible channels—there are both water-soluble and membrane-spanning examples. Recent computational designs of water-soluble α-helical barrels with five to seven helices have advanced the design field considerably. Here we identify and classify analogous and more complicated membrane-spanning α-helical barrels from the Protein Data Bank. These provide tantalizing but tractable targets for protein engineering and de novo protein design.


ISAMBARD: an open-source computational environment for biomolecular analysis, modelling and design

Christopher W. Wood, Jack W. Heal, Andrew R. Thomson, Gail J. Bartlett, Amaurys A. Ibarra, R. Leo Brady, Richard B. Sessions and Derek N. Woolfson

Bioinformatics 2017 doi: 10.1093/bioinformatics/btx352

Motivation: The rational design of biomolecules is becoming a reality. However, further computational tools are needed to facilitate and accelerate this, and to make it accessible to more users. Results: Here we introduce ISAMBARD, a tool for structural analysis, model building and rational design of biomolecules. ISAMBARD is open-source, modular, computationally scalable and intuitive to use. These features allow non-experts to explore biomolecular design in silico. ISAMBARD addresses a standing issue in protein design, namely, how to introduce backbone variability in a controlled manner. This is achieved through the generalisation of tools for parametric modelling, describing the overall shape of proteins geometrically, and without input from experimentally determined structures. This will allow backbone conformations for entire folds and assemblies not observed in nature to be generated de novo, that is, to access the ‘dark matter of protein-fold space’. We anticipate that ISAMBARD will find broad applications in biomolecular design, biotechnology and synthetic biology.


Engineering protein stability with atomic precision in a monomeric miniprotein

Emily G. Baker, Christopher Williams, Kieran L. Hudson, Gail J. Bartlett, Jack W. Heal, Kathryn L. Porter Goff, Richard B. Sessions, Matthew P. Crump & Derek N. Woolfson

Nature Chemical Biology 2017 doi:10.1038/nchembio.2380

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPalpha-Tyr, a designed peptide comprising an alpha-helix buttressed by a polyproline II helix. PPalpha-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (TM) of 39 °C. NMR structures of PPalpha-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPalpha is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the TM to 20 °C. We attribute this result to the loss of CH-pi interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-pi interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.


Coiled-Coil Design: Updated and Upgraded

Derek N. Woolfson

Subcellular Biochemistry 2017 82:35-61. In Fibrous Proteins: Structures and Mechanisms Eds D. A. D Parry and J. M. Squire, Springer. DOI:10.1007/978-3-319-49674-0_2

Alpha-Helical coiled coils are ubiquitous protein-folding and protein-interaction domains in which two or more alpha-helical chains come together to form bundles. Through a combination of bioinformatics analysis of many thousands of natural coiled-coil sequences and structures, plus empirical protein engineering and design studies, there is now a deep understanding of the sequence-to-structure relationships for this class of protein architecture. This has led to considerable success in rational design and what might be termed in biro de novo design of simple coiled coils, which include homo- and hetero-meric parallel dimers, trimers and tetramers. In turn, these provide a toolkit for directing the assembly of both natural proteins and more complex designs in protein engineering, materials science and synthetic biology. Moving on, the increased and improved use of computational design is allowing access to coiled-coil structures that are rare or even not observed in nature, for example alpha-helical barrels, which comprise five or more α-helices and have central channels into which different functions may be ported. This chapter reviews all of these advances, outlining improvements in our knowledge of the fundamentals of coiled-coil folding and assembly, and highlighting new coiled coil-based materials and applications that this new understanding is opening up. Despite considerable progress, however, challenges remain in coiled-coil design, and the next decade promises to be as productive and exciting as the last.


N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Jordan M. Fletcher, Gail J. Bartlett, Aimee L. Boyle, Jonathan J. Danon, Laura E. Rush, Andrei N. Lupas, and Derek N. Woolfson

ACS Chemical Biology 2016 DOI:10.1021/acschembio.6b00935

The alpha-helical coiled coil is one of the best-studied protein–protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix–helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.


A monodisperse transmembrane alpha-helical peptide barrel

Kozhinjampara R. Mahendran, Ai Niitsu, Lingbing Kong, Andrew R. Thomson, Richard B. Sessions, Derek N. Woolfson and Hagan Bayley

Nature Chemistry 2016 DOI:10.1038/nchem.2647

The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid alpha-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such alpha-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.


Installing hydrolytic activity into a completely de novo protein framework

Antony J. Burton, Andrew R. Thomson, William M. Dawson, R. Leo Brady & Derek N. Woolfson

Nature Chemistry 2016 DOI:10.1038/nchem.2555

The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.


Controlling the Assembly of Coiled-Coil Peptide Nanotubes

Franziska Thomas, Natasha C. Burgess, Andrew R. Thomson and Derek N. Woolfson

Angewandte Chemie International Edition 2016 55:987-991 DOI: 10.1002/anie.201509304

An ability to control the assembly of peptide nanotubes (PNTs) would provide biomaterials for applications in nanotechnology and synthetic biology. Recently, we presented a modular design for PNTs using a-helical barrels with tunable internal cavities as building blocks. These first-generation designs thicken beyond single PNTs. Herein we describe strategies for controlling this lateral association, and also for the longitudinal assembly. We show that PNT thickening is pH sensitive, and can be reversed under acidic conditions. Based on this, repulsive charge interactions are engineered into the building blocks leading to the assembly of single PNTs at neutral pH. The building blocks are modified further to produce covalently linked PNTs via native chemical ligation, rendering ca. 100 nm-long nanotubes. Finally, we show that small molecules can be sequestered within the interior lumens of single PNTs.


On the satisfaction of backbone-carbonyl lone pairs of electrons in protein structures

Gail J. Bartlett and Derek N. Woolfson

Protein Science 2016 25:887-897 DOI: 10.1002/pro.2896

Protein structures are stabilized by a variety of noncovalent interactions (NCIs), including the hydrophobic effect, hydrogen bonds, electrostatic forces and van der Waals’ interactions. Our knowledge of the contributions of NCIs, and the interplay between them remains incomplete. This has implications for computational modeling of NCIs, and our ability to understand and predict protein structure, stability, and function. One consideration is the satisfaction of the full potential for NCIs made by back-bone atoms. Most commonly, backbone-carbonyl oxygen atoms located within a-helices and b-sheets are depicted as making a single hydrogen bond. However, there are two lone pairs of electrons to be satisfied for each of these atoms. To explore this, we used operational geometric definitions to generate an inventory of NCIs for backbone-carbonyl oxygen atoms from a set of high-resolution protein structures and associated molecular-dynamics simulations in water. We included more-recently appreciated, but weaker NCIs in our analysis, such as n->pi* interactions, Ca-H bonds and methyl-H bonds. The data demonstrate balanced, dynamic systems for all proteins, with most backbone-carbonyl oxygen atoms being satisfied by two NCIs most of the time. Combinations of NCIs made may correlate with secondary structure type, though in subtly different ways from traditional models of a- and b-structure. In addition,we find examples of under- and over-satisfied carbonyl-oxygen atoms, and we identify both sequence-dependent and sequence-independent secondary-structural motifs in which these reside. Our analysis provides a more-detailed understanding of these contributors to protein structure and stability, which will be of use in protein modeling, engineering and design.


Carbohydrate-aromatic Interactions in Proteins

Kieran L. Hudson, Gail J. Bartlett, Roger C. Diehl, Jon Agirre, Timothy Gallagher, Laura L. Kiessling and Derek N. Woolfson

Journal of the American Chemical Society 2015 in press DOI: 10.1021/jacs.5b08424

Protein-carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C-H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C-H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C-H bonds engage more often in CH-pi interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate-aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C-H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein-carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein-carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.


Modular Design of Self-Assembling Peptide-Based Nanotubes

Natasha C. Burgess, Thomas H. Sharp, Franziska Thomas, Christopher W. Wood, Andrew R. Thomson, Nathan R. Zaccai, R. Leo Brady, Louise C. Serpell and Derek N. Woolfson

Journal of the American Chemical Society 2015 137 (33) pp 10554-10562 DOI: 10.1021/jacs.5b03973

An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended alpha-helical barrels, that is, preassembled bundles of alpha-helices with central channels, can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric alpha-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction, and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric, and heptameric alpha-helical barrels sequester hydrophobic dye within their lumens.


De novo protein design: How do we expand into the universe of possible protein structures?

Derek N. Woolfson, Gail J. Bartlett, Antony J. Burton, Jack W. Heal, Ai Niitsu, Andrew R. Thomson and Christopher W. Wood

Current Opinion in Structural Biology 2015 DOI: 10.1016/

Protein scientists are paving the way to a new phase in protein design and engineering. Approaches and methods are being developed that could allow the design of proteins beyond the confines of natural protein structures. This possibility of designing entirely new proteins opens new questions: What do we build? How do we build into protein-structure space where there are few, if any, natural structures to guide us? To what uses can the resulting proteins be put? And, what, if anything, does this pursuit tell us about how natural proteins fold, function and evolve? We describe the origins of this emerging area of fully de novo protein design, how it could be developed, where it might lead, and what challenges lie ahead.


Functionalized Alpha-helical Peptide Hydrogels for Neural Tissue Engineering

Nazia Mehrban, Bangfu Zhu, Francesco Tamagnini, Fraser I. Young, Alexandra Wasmuth, Kieran L. Hudson, Andrew R. Thomson, Martin A. Birchall, Andrew D. Randall, Bing Song, and Derek N. Woolfson

ACS Biomaterials Science & Engineering 2015 DOI: 10.1021/acsbiomaterials.5b00051

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair


Local and macroscopic electrostatic interactions in single alpha-helices

Emily G. Baker, Gail J. Bartlett, Matthew P. Crump, Richard B. Sessions, Noah Linden, Charl F. J. Faul and Derek N. Woolfson

Nature Chemical Biology 2015 11, pp 221-228 DOI: 10.1038/nchembio.1739

The noncovalent forces that stabilize protein structures are not fully understood. One way to address this is to study equilibria between unfolded states and alpha-helices in peptides. Electrostatic forces - which include interactions between side chains, the backbone and side chains, and side chains and the helix macrodipole - are believed to contribute to these equilibria. Here we probe these interactions experimentally using designed peptides. We find that both terminal backbone-side chain and certain side chain-side chain interactions (which include both local effects between proximal charges and interatomic contacts) contribute much more to helix stability than side chain-helix macrodipole electrostatics, which are believed to operate at larger distances. This has implications for current descriptions of helix stability, the understanding of protein folding and the refinement of force fields for biomolecular modeling and simulations. In addition, this study sheds light on the stability of rod-like structures formed by single alpha-helices, which are common in natural proteins such as non-muscle myosins.


Construction and Characterization of Kilobasepair Densely Labeled Peptide-DNA

Suzana Kovacic, Laleh Samii, Guillaume Lamour, Hongbin Li, Heiner Linke, Elizabeth H. C. Bromley, Derek N. Woolfson, Paul M. G. Curmi, and Nancy R. Forde

Biomacromolecules 2014 15 (11), pp 4065-4072 DOI: 10.1021/bm501109p

Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.


Computational design of water-soluble alpha-helical barrels

Andrew R Thomson, Christopher W Wood, Antony J Burton, Gail J Bartlett, Richard B Sessions, R Leo Brady & Derek N Woolfson

Science 2014 Vol 346:485-488 DOI: 10.1126/science.1257452

The design of protein sequences that fold into prescribed de novo structures is challenging. General solutions to this problem require geometric descriptions of protein folds and methods to fit sequences to these. The alpha-helical coiled coils present a promising class of protein for this and offer considerable scope for exploring hitherto unseen structures. For alpha-helical barrels, which have more than four helices and accessible central channels, many of the possible structures remain unobserved. Here, we combine geometrical considerations, knowledge-based scoring, and atomistic modeling to facilitate the design of new channel-containing alpha-helical barrels. X-ray crystal structures of the resulting designs match predicted in silico models. Furthermore, the observed channels are chemically defined and have diameters related to oligomer state, which present routes to design protein function.


Synthetic Biology: Writing the future with biomolecules

Derek N Woolfson

Science in Parliament 2014

Synthetic biology is an emerging field that aims to make the engineering of biology easier, more reliable and more predictable. It combines understanding and methods from the biological and physical sciences with engineering principles and approaches. It is a truly multidisciplinary endeavour that requires input from experimental scientists, theoreticians, engineers and social scientists to succeed. If it takes root, the promises are considerable, and synthetic biology will have an impact on how we think about basic research in the biological sciences through to how we exploit it in the biotech, pharma and agrichem sectors. Through coordinated efforts from government, the Research Councils, industry and the academic research community over the past 7 years, the UK has built an extremely strong base for synthetic-biology research. This is largely founded in the universities and basic-research facilities, but there are strong links with industry. The challenges ahead are to grow this base to deliver high-quality basic science, which, in turn, will lead to applications underpinning UK SMEs and industry.


CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil-protein assemblies

Christopher W Wood, Marc Bruning, Amaurys Á Ibarra, Gail J Bartlett, Andrew R Thomson, Richard B Sessions, R Leo Brady, and Derek N Woolfson

Bioinformatics 2014 30 (21): 3029-3035. DOI:10.1093/bioinformatics/btu502

Motivation: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably, and to design proteins de novo. Homology based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires: mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. Results: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the a-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks, and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allow users to build and assess models, in which helix geometry, coiled-coil architecture and topology, and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types; and generating models for rarer and de novo coiled-coil structures. Availability: CCBuilder is freely available, without registration, at


Assessing Cellular Response to Functionalized a-Helical Peptide Hydrogels

Nazia Mehrban, Edgardo Abelardo, Alexandra Wasmuth, Kieran L. Hudson, Leanne M. Mullen, Andrew R. Thomson, Martin A. Birchall and Derek N. Woolfson

Advanced Healthcare Materials 2014 3:1387-1391 DOI: 10.1002/adhm.201400065


A Catalytic Role For Methionine Revealed By A Combination Of Computation And Experiments On Phosphite Dehydrogenase

Kara E. Ranaghan, John E. Hung, Gail J. Bartlett, Tiddo J. Mooibroek, Jeremy N. Harvey, Derek N. Woolfson, Wilfred A. van der Donk and Adrian J. Mulholland

Chemical Science (5) 2191-21992014 DOI:

Combined quantum mechanics/molecular mechanics (QM/MM) simulations of the reaction catalysed by phosphite dehydrogenase (PTDH) identify Met53 as important for catalysis. This catalytic role is verified by experiments (including replacement by norleucine and selenomethionine), which show that mutation of this residue significantly affects kcat, without changing KM for phosphite. QM/MM and ab initio QM calculations show that the catalytic effect is electrostatic in nature. The side chain of Met53 specifically stabilizes the transition state for the hydride transfer step of the reaction catalysed by PTDH, forming a ‘face-on’ interaction with His292. To our knowledge, a defined catalytic role for methionine in an enzyme (as opposed to a steric or binding effect, or interaction with a metal ion) has not previously been identified. Analyses of the Protein Data Bank and Cambridge Structural Database indicate that this type of interaction may be relatively widespread, with implications for enzyme-catalysed reaction mechanisms and protein structure.


Signatures of n->pi* Interactions in Proteins

Robert W. Newberry, Gail J. Bartlett, Brett VanVeller, Derek N. Woolfson & Ronald T. Raines

Protein Science 2014 23:284-288 DOI:

The folding of proteins is directed by a variety of interactions, including hydrogen bonding, electrostatics, van der Waals' interactions, and the hydrophobic effect. We have argued previously that an n->pi* interaction between carbonyl groups be added to this list. In an n->pi* interaction, the lone pair (n) of one carbonyl oxygen overlaps with the pi* antibonding orbital of another carbonyl group. The tendency of backbone carbonyl groups in proteins to engage in this interaction has consequences for the structures of folded proteins that we unveil herein. First, we employ density functional theory to demonstrate that the n->pi* interaction causes the carbonyl carbon to deviate from planarity. Then, we detect this signature of the n->pi* interaction in high-resolution structures of proteins. Finally, we demonstrate through natural population analysis that the n->pi* interaction causes polarization of the electron density in carbonyl groups and detect that polarization in the electron density map of cholesterol oxidase, further validating the existence of n->pi* interactions. We conclude that the n->pi* interaction is operative in folded proteins.


Interplay of Hydrogen Bonds and n->pi* Interactions in Proteins

Gail J. Bartlett, Robert W. Newberry, Brett VanVeller, Ronald T. Raines, Derek N. Woolfson

J Am Chem Soc 135 (49) pp 18682–18688 2013 DOI:

Protein structures are stabilized by multiple weak interactions, including the hydrophobic effect, hydrogen bonds, electrostatic effects, and van der Waals interactions. Among these interactions, the hydrogen bond is distinct in having its origins in electron delocalization. Recently, another type of electron delocalization, the n->pi* interaction between carbonyl groups, has been shown to play a role in stabilizing protein structure. Here we examine the interplay between hydrogen bonding and n->pi* interactions. To address this issue, we used data available from high-resolution protein crystal structures to interrogate asparagine side-chain oxygen atoms that are both acceptors of a hydrogen bond and donors of an n->pi* interaction. Then we employed natural bond orbital analysis to determine the relative energetic contributions of the hydrogen bonds and n->pi* interactions in these systems. We found that an n->pi* interaction is worth ~5-25% of a hydrogen bond and that stronger hydrogen bonds tend to attenuate or obscure n->* interactions. Conversely, weaker hydrogen bonds correlate with stronger n->pi* interactions and demixing of the orbitals occupied by the oxygen lone pairs. Thus, these two interactions conspire to stabilize local backbone-side-chain contacts, which argues for the inclusion of n->pi* interactions in the inventory of non-covalent forces that contribute to protein stability and thus in force fields for biomolecular modeling.


Accessibility, Reactivity, and Selectivity of Side Chains within a Channel of de Novo Peptide Assembly

Antony J. Burton, Franziska Thomas, Christopher Agnew, Kieran L. Hudson, Stephen E. Halford, R. Leo Brady, Derek N. Woolfson

J Am Chem Soc 135:12524-12527 2013 DOI:

Ab initio design of enzymes requires precise and predictable positioning of reactive functional groups within accessible and controlled environments of de novo protein scaffolds. Here we show that multiple thiol moieties can be placed within a central channel, with approximate dimensions 6 ×42 Å of a de novo, six-helix peptide assembly (CC-Hex). Layers of six cysteine residues are introduced at two different sites ~6 (the "L24C" mutant) and ~17 Å(L17C) from the C-terminal opening of the channel. X-ray crystal structures confirm the mutant structures as hexamers with internal free thiol, rather than disulfide-linked cysteine residues. Both mutants are hexa-alkylated upon addition of iodoacetamide, demonstrating accessibility and full reactivity of the thiol groups. Comparison of the alkylation and unfolding rates of the hexamers indicates that access is directly through the channel and not via dissociation and unfolding of the assembly. Moreover, neither mutant reacts with iodoacetic acid, demonstrating selectivity of the largely hydrophobic channel. These studies show that it is possible to engineer reactive side chains with both precision and control into a de novo scaffold to produce protein-like structures with chemoselective reactivity.


Prediction and analysis of higher-order coiled-coils: Insights from proteins of the extracellular matrix, tenascins and thrombospondins

Thomas L. Vincent, Derek N. Woolfson, Josephine C. Adams

International Journal of Biochemistry and Cell Biology 2013 DOI: 10.1016/j.biocel.2013.07.011

Alpha-Helical coiled-coil domains (CCDs) direct protein oligomerisation in many biological processes and are of great interest as tools in protein engineering. Although CCDs are recognizable from protein sequences, prediction of oligomer state remains challenging especially for trimeric states and above. Here we evaluate LOGICOIL, a new multi-state predictor for CCDs, with regard to families of extracellular matrix proteins. Tenascins, which are known to assemble as trimers, were the first test case. LOGICOIL out-performed other algorithms in predicting trimerisation of these proteins and sequence analyses identified features associated with many other trimerising CCDs. The thrombospondins are a larger and more ancient family that includes sub-groups that assemble as trimers or pentamers. LOGICOIL predicted the pentamerising CCDs accurately. However, prediction of TSP trimerisation was relatively poor, although accuracy was improved by analyzing only the central regions ofthe CCDs. Sequence clustering and phylogenetic analyses grouped the TSP CCDs into three clades comprising trimers and pentamers from vertebrates, and TSPs from invertebrates. Sequence analyses revealed distinctive, conserved features that distinguish trimerising and pentamerising CCDs. Together, these analyses provide insight into the specification of higher-order CCDs that should direct improved CCD predictions and future experimental investigations of sequence-to-structure functional relationships.


Controlled microfluidic switching in arbitrary time-sequences with low drag

Cassandra S. Niman, Jason P. Beech, Jonas O. Tegenfeldt, Paul M. G. Curmi, Derek N. Woolfson, Nancy R. Forde, Heiner Linke

Lab on a Chip 2013 DOI: 10.1039/c3lc50194a

The ability to test the response of cells and proteins to a changing biochemical environment is of interest for studies of fundamental cell physiology and molecular interactions. In a common experimental scheme the cells or molecules of interest are attached to a surface and the composition of the surrounding fluid is changed. It is desirable to be able to switch several different biochemical reagents in any arbitrary order, and to keep the flow velocity low enough so that the cells and molecules remain attached and can be expected to retain their function. Here we develop a device with these capabilities, using U-shaped access channels. We use total-internal reflection fluorescence microscopy to characterize the time-dependent change in concentration during switching of solutions near the device surface. Well-defined fluid interfaces are formed in the immediate vicinity of the surface ensuring distinct switching events. We show that the experimental data agrees well with Taylor.Aris theory in its range of validity. In addition, we find that well-defined interfaces are achieved also in the immediate vicinity of the surface, where analytic approaches and numerical models become inaccurate. Assisted by finite-element modelling, the details of our device were designed for use with a specific artificial protein motor, but the key results are general and can be applied to a wide range of biochemical studies in which switching is important.


Synthetic Biology goes live

Derek N. Woolfson

The Biochemist 2013

On 14 November last year, the Biochemical Society, the Royal Society of Chemistry, the think-tank BioCentre and the University of Bristol co-hosted a debate on synthetic biology, which was webcast live. Dek Woolfson co-chaired the event from Bristol. Here are his reflections and conclusions from the evening, including some advice on how we might approach the broader issues of the subject and events like this in the future


Self-Assembling Cages from Coiled-Coil Peptide Modules

Jordan M. Fletcher; Robert L. Harniman; Frederick R. H. Barnes; Aimee L. Boyle; Andrew Collins; Judith Mantell; Thomas H. Sharp; Massimo Antognozzi; Paula J. Booth; Noah Linden; Mervyn J. Miles; Richard B. Sessions; Paul Verkade; Derek N. Woolfson

Science 2013 340, 595-599 DOI: 10.1126/science.1233936
News and views of the above article can be found here: Review 1 Review 2 Review 3

An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals, and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back-to-back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.


A Set of de Novo Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime

Franziska Thomas; Aimee L. Boyle; Antony J. Burton; Derek N. Woolfson

J Am Chem Soc 2013 DOI: 10.1021/ja312310g

The availability of peptide and protein components that fold to defined structures with tailored stabilities would facilitate rational protein engineering and synthetic biology. We have begun to generate a toolkit of such components based on de novo designed coiled-coil peptides that mediate protein.protein interactions. Here, we present a set of coiled-coil heterodimers to add to the toolkit. The lengths of the coiled-coil regions are 21, 24, or 28 residues, which deliver dissociation constants in the micromolar to sub-nanomolar range. In addition, comparison of two related series of peptides highlights the need for including polar residues within the hydrophobic interfaces, both to specify the dimer state over alternatives and to fine-tune the dissociation constants.


LOGICOIL - Multi-state prediction of coiled-coil oligomeric state

Thomas L. Vincent; Peter J. Green; Derek N. Woolfson

Bioinformatics 2013 29(1):69-76 DOI: 10.1093/bioinformatics/bts648

Motivation: The coiled coil is a ubiquitous alpha-helical protein-structure domain that directs and facilitates protein-protein interactions in a wide variety of biological processes. At the protein-sequence level, the coiled coil is readily recognized via a conspicuous heptad repeatof hydrophobic and polar residues. However, structurally coiled coils are more complicated, existing in a wide range of oligomer states and topologies. As a consequence, predicting these various states from sequence remains an unmet challenge. Results: This work introduces LOGICOIL, the ?rst algorithm to address the problem of predicting multiple coiled-coil oligomeric states from protein-sequence information alone. By covering \> 90% of the known coiled-coil structures, LOGICOIL is a net improvement compared to other existing methods, which achieve a predictive coverage of approximately 31% of this population. This leap in predictive power offers better opportunities for genome-scale analysis, and analyses of coiled-coil containing protein assemblies. Availability: LOGICOIL is available via a web-interface at Source code, training sets and Supporting Information can be downloaded from the same site.


Strong Contributions from Vertical Triads to Helix-Partner Preferences in Parallel Coiled Coils

Steinkruger, Jay; Bartlett, Gail; Woolfson, Derek N.; Gellman, Samuel

J. Am. Chem. Soc. 2012 134(38):15652-5

coming soon


Squaring the Circle in Peptide Assembly: From Fibers to Discrete Nanostructures by De Novo Design

Aimee L. Boyle , Elizabeth H. C. Bromley , Gail J Bartlett , Richard B Sessions , Thomas H Sharp , Claire L Williams , Paul M. G. Curmi , Nancy R Forde , Heiner Linke , and Derek N. Woolfson

J. Am. Chem. Soc. 2012 134(37):15457-67

The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide- and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represent a new route for constructing complex peptide-based assemblies and biomaterials.


Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design

Thomas H. Sharp, Marc Bruning, Judith Mantell, Richard B. Sessions,Andrew R. Thomson, Nathan R. Zaccai, R. Leo Brady, Paul Verkade,and Derek N. Woolfson

Proc. Natl. Acad. Sci. U. S. A. 2012 109 13266-71 (2012)

Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.


A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology

Jordan M. Fletcher, Aimee L. Boyle, Marc Bruning, Gail J. Bartlett, Thomas L. Vincent, Nathan R. Zaccai, Craig T. Armstrong, Elizabeth H. C. Bromley, Paula J. Booth, R. Leo Brady, Andrew R. Thomson, and Derek N. Woolfson

ACS Synthetic Biology 2012 6 240-250 (2012)

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.


New currency for old rope: from coiled-coil assemblies to a-helical barrels

Derek N Woolfson, Gail J Bartlett, Marc Bruning, and Andrew R Thomson

Current Opinion in Structural Biology 2012 432-441 (2012)

a-Helical coiled coils are ubiquitous protein–protein-interaction domains. They share a relatively straightforward sequence repeat, which directs the folding and assembly of amphipathic a- helices. The helices can combine in a number of oligomerisation states and topologies to direct a wide variety of protein assemblies. Although in nature parallel dimers, trimers and tetramers dominate, the potential to form larger oligomers and more-complex assemblies has long been recognised. In particular, complexes above pentamer are interesting because they are barrel-like, having central channels or pores with well- defined dimensions and chemistry. Recent empirical and rational design experiments are beginning to chart this potential new territory in coiled-coil space, leading to intriguing new structures, and possibilities for functionalisation and applications.


Rational Design of Peptide-Based Biosupramolecular Systems

Aimee L. Boyle and Derek N. Woolfson

Supramolecular Chemistry: From Molecules To Nanomaterials 2012 1639 - 1664 (2012)

A highly attractive feature of peptides and proteins for use in what might be termed “biosupramolecular chemistry” is their ability to self-assemble in water with exquisite speci- ficity and high affinities through noncovalent interactions. An aim of peptide design and engineering is to exploit this to create new supramolecular assemblies from the bottom up.1, 2 Within this, one approach is to program simple pep- tide and protein building blocks (which might be referred to as “tectons”) to fold and self-associate in prescribed ways. This de novo design route contrasts with top-down approaches, which are concerned with protein engineering and the modification of already existing and even assembled natural proteins. The bottom-up approach has the potential advantage that it allows for full design and control over folding, assembly, size, and shape of the targets, and so opens up a wide range of structural space. It also tests our understanding of peptide and protein folding and assembly directly. However, at present, it has clear disadvantages over engineering natural systems, as we do not fully under- stand protein folding, and, therefore, the rational design of protein structure and function is in its infancy.3, 4 This chapter focuses on supramolecular assemblies that are formed using a variety of de novo designed peptide- based tectons. A brief introduction to amino acids (the building blocks of peptides and proteins) is given, followed by a discussion of the basic structures that polypeptide chains of amino acids can adopt. These structures form the basis of the supramolecular assemblies that will be reviewed. The subsequent sections provide details of recent examples of repetitive, effectively “infinite,” and discrete supramolecular peptide-based assemblies, and also a dis- cussion of their potential applications.


Design and Construction of a One-Dimensional DNA Track for an Artificial Molecular Motor.

Suzana Kovacic, Laleh Samii, Derek N. Woolfson, Paul M. G. Curmi, Heiner Linke, Nancy R. Forde, and Gerhard A. Blab

Journal of Nanomaterials 2012 1-10 (2012)

DNA is a versatile heteropolymer that shows great potential as a building block for a diverse array of nanostructures. We present here a solution to the problem of designing and synthesizing a DNA-based nanostructure that will serve as the track along which an artificial molecular motor processes. This one-dimensional DNA track exhibits periodically repeating elements that provide specific binding sites for the molecular motor. Besides these binding elements, additional sequences are necessary to label specific regions within the DNA track and to facilitate track construction. Designing an ideal DNA track sequence presents a particular challenge because of the many variable elements that greatly expand the number of potential sequences from which the ideal sequence must be chosen. In order to find a suitable DNA sequence, we have adapted a genetic algorithm which is well suited for a large but sparse search space. This algorithm readily identifies long DNA sequences that include all the necessary elements to both facilitate DNA track construction and to present appropriate binding sites for the molecular motor. We have successfully experimentally incorporated the sequence identified by the algorithm into a long DNA track meeting the criteria for observation of the molecular motor's activity.


The d'--d--d' Vertical Triad Is Less Discriminating Than the a'--a--a' Vertical Triad in the Antiparallel Coiled-Coil Dimer Motif.

Steinkruger JD, Bartlett GJ, Hadley EB, Fay L, Woolfson DN, Gellman SH

Journal of the American Chemical Society 134(5) 2626-2633 (2012) DOI: 10.1021/ja208855x

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.


Metallopolymer-Peptide Hybrid Materials: Synthesis and Self-Assembly of Functional, Polyferrocenylsilane-Tetrapeptide Conjugates.

Tangbunsuk S, Whittell GR, Ryadnov MG, Vandermeulen GW, Woolfson DN, Manners I

Chemistry doi: 10.1002/chem.201102223Jan 19 (2012) DOI: 10.1002/chem.201102223

Conjugates of poly(ferrocenyldimethylsilane) (PFDMS) with Ac-(GA)(2) -OH, Ac-A(4)-OH, Ac-G(4) -OH and Ac-V(4) -OH have been prepared by reaction of the tetrapeptide units with the amino-terminated metallopolymer. The number average degree of polymerisation (DP(n) ) of the PFDMS was approximately 20 and comparable materials with shorter (DP(n) ≈10) and/or amorphous chains have been prepared by the same procedure. Poly(ferrocenylethylmethylsilane) (PFEMS) was employed for the latter purpose. All conjugates were characterised by GPC, MALDI-TOF MS, NMR and IR spectroscopy. With the exception of Ac-V(4) -PFDMS(20) , all materials exhibited some anti-parallel β-sheet structure in the solid state. The self-assembly of the conjugates was studied in toluene by DLS. The vast majority of the materials, irrespective of peptide sequence or chain crystallinity, afforded fibres consisting of a peptidic core surrounded by a PFS corona. These fibres were found in the form of cross-linked networks by TEM and AFM. The accessibility of the chemically reducing PFS corona has been demonstrated by the localised formation of silver nanoparticles on the surface of the fibres.


Tuning the performance of an artificial protein motor

NJ Kuwada, MJ Zuckermann, EH Bromley, RB Sessions, PM Curmi, NR Forde, DN Woolfson, H Linke

PHYSICAL REVIEW E 84(3) 031922 (2011) DOI: 10.1103/PhysRevE.84.031922

The Tumbleweed (TW) is a concept for an artificial, tri-pedal, protein-based motor designed to move unidirectionally along a linear track by a diffusive tumbling motion. Artificial motors offer the unique opportunity to explore how motor performance depends on design details in a way that is open to experimental investigation. Prior studies have shown that TW's ability to complete many successive steps can be critically dependent on the motor's diffusional step time. Here, we present a simulation study targeted at determining how to minimize the diffusional step time of the TW motor as a function of two particular design choices: nonspecific motor-track interactions and molecular flexibility. We determine an optimal nonspecific interaction strength and establish a set of criteria for optimal molecular flexibility as a function of the nonspecific interaction. We discuss our results in the context of similarities to biological, linear stepping diffusive molecular motors with the aim of identifying general engineering principles for protein motors.


A de novo peptide hexamer with a mutable channel

NR Zaccai, B Chi, AR Thomson, AL Boyle, GJ Bartlett, M Bruning, N Linden, RB Sessions, PJ Booth, RL Brady and DN Woolfson

Nature Chemical Biology 7 935-941 (2011) DOI: 10.1038/NCHEMBIO.692
News and reviews on the topic of this paper can be found at the following links: Review 1

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6 Å channel is permeable to water. One layer of leucine residues within the channel is mutable, accepting polar aspartic acid and histidine side chains, which leads to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)3 heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.


Random-Coil: α-Helix Equilibria as a Reporter for the LewisX-LewisX Interaction

TM Altamore, C Fernandez-Garcia, AH Gordon, T Hubscher, N Promsawan, MG Ryadnov, AJ Doig, DN Woolfson and T Gallagher

Angewandte Chemie 50(47)11167-11171 (2011) DOI: 10.1002/anie.201101055

Probing weak interactions: A peptide random-coil α-helix equilibrium has been used to identify a weak carbohydrate-carbohydrate interaction (CCI). Glucose and lactose destabilized the helical conformer while LewisX trisaccharide led to increased helicity. Though small, the trend observed indicates that this peptide reporter can detect a single CCI in isolation.


SCORER 2.0: An algorithm for distinguishing parallel dimeric and trimeric coiled-coil sequences

CR Armstrong, TL Vincent, PJ Green and DN Woolfson

Bioinformatics 27(14)1908-1914 (2011) DOI: 10.1093/bioinformatics/btr299

The coiled coil is a ubiquitous α-helical protein-structure domain that directs and facilitates protein-protein interactions in a wide variety of biological processes. At the protein-sequence level, coiled coils are quite straightforward and readily recognised via the conspicuous heptad repeats of hydrophobic and polar residues. However, structurally they are more complicated, existing in a range of oligomer states and topologies. Here we address the issue of predicting coiled-coil oligomeric state from protein sequence. The predominant coiled-coil oligomer states in Nature are parallel dimers and trimers. Here we improve and retrain the first-published algorithm, SCORER, that distinguishes these states, and test it against the current standard, MultiCoil. The SCORER algorithm has been revised in two key respects: First, the statistical basis for SCORER is improved markedly. Second, the training set for SCORER has been expanded and updated to include only structurally validated coiled coils. The result is a much-improved oligomer-state predictor that outperforms MultiCoil, particularly in assigning oligomer state to short coiled coils, and those that are diverse from the training set. SCORER 2.0 is available via a web-interface at Source code, training sets and Supporting Information can be downloaded from the same site.


Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a Gram-negative bacterium.

X Xiong, EHC Bromley, P Oelschlaeger, DN Woolfson and J Spencer

Nucleic Acids Research 39, 9, 3917-3927 (2011) DOI: 10.1093/nar/gkq1296

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in Gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for Gram-negative Qnr proteins. The AhQnr crystal structure, the first for any Gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of Gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A ‘tower’ and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in Gram-negative bacteria.


Designed coiled coils promote folding of a recombinant bacterial collagen.

A Yoshizumi, JM Fletcher, Z Yu, A Persikov, GJ Bartlett, AL Boyle, TL Vincent, DN Woolfson and B Brodsky

J. Biol. Chem. 286, 20, 1715-1720 (2011)

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the S. pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical occurs N-terminal to the collagen triple-helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, 3-stranded, α-helical coiled coil (CC), either at the N-terminus (CC-CL) or the C-terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 ℃ and CC > 90 ℃. Heating the hybrid proteins to 50 ℃ unfolds CL leaving CC intact, and upon cooling the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 ℃ thermal stability for CL, but shows less triple-helix at low temperature and less denaturation at 50 ℃. Most strikingly however, in CC-CL-CC the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end; whereas, initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this, since, where present, there is generally only one coiled-coil domain close to the triple-helix and it is nearly always N-terminal to the collagen repeat.


De novo designed peptides for biological applications.

AL Boyle and DN Woolfson

Chem. Soc. Rev., 40, 4295-4306 (2011)

In recent years our ability to design and assemble peptide-based materials and objects de novo (i.e. from first principles) has improved considerably. This brings us to a point where the resulting assemblies are quite sophisticated and amenable to engineering in new functions. Whilst such systems could be used in a variety of ways, biological applications are of particular interest because of the demand for biocompatible, readily produced systems with potential as drug-delivery agents, components of biosensors and scaffolds for 3D cell and tissue culture. This tutorial review describes the building blocks (or tectons) that are being used in peptide assembly, highlights a range of materials and objects that have been produced, notably hydrogels and virus-like particles, and introduces a number of potential applications for the designs.


Polyelectrolyte-surfactant nanocomposite membranes formed at a liquid-liquid interface.

DB Carew, KJ Channon, I Manners, and DN Woolfson

Soft Matter 7 3475-3481 (2011)

There are three main methods for constructing polyelectrolyte-surfactant membranes at interfaces, and all require solid supports. However, a recent paper demonstrates that peptide amphiphiles and a biological polyelectrolyte can form membranes at the water-water interface. Here, we show that similarly unsupported, columnar membranes can be achieved readily with commercially available polyelectrolytes and surfactants. We demonstrate a new preparation process, and that these membranes provide suitable substrates for silica deposition to render columnar, free-standing silica replicas. In addition, we introduce a new, high-throughput, combinatorial method for studying and optimizing membrane formation.


Computer simulations of the growth of synthetic peptide fibres.

TP Stedall, MF Butler, DN Woolfson, and S Hanna

Eur. Phys. J. E 34(5) (2011)
This work was illustrated on the cover of the European Physical Journal E - Soft Matter and Biological Physics. Click here to see the cover.

We present a coarse-grained computer model designed tostudy the growth of fibres in a synthetic self-assembling peptide system. The system consists of two 28 residue α-helical sequences, denoted AB and CD, in which the interactions between the half peptides, A, B, C and D, may be tuned individually to promote different types of growth behaviour. In the model, AB and CD are represented by double ended rods, with interaction sites distributed along their lengths. Monte Carlo simulations are performed to follow fibre growth. It is found that lateral and longitudinal growth of the fibre are governed by different mechanisms -the former is diffusion limited with a very small activation energy for the addition of units, whereas the latter occurs via a process of secondary nucleation at the fibre ends. As a result, longitudinal growth generally proceeds more slowly than lateral growth. Furthermore, it is shown that the aspect ratio of the growing fibre may be controlled by adjusting the temperature and the relative strengths of the interactions. The predictions of the model are discussed in the context of published data from real peptide systems.


Synthetic Biology: a bit of rebranding, or something new and inspiring?

DN Woolfson and EHC Bromley

The Biochemist 33 19-25 (2011)

A new approach in biology is emerging and gathering a broad band of advocates. Synthetic biology aims to improve our abilities to engineer biological molecules, assemblies and systems; to design and develop biomimetic systems; and to apply these to useful ends. It brings together the biological and physical sciences, applying engineering and mathematical principles. Currently, a number of different approaches are being explored in synthetic biology, which are meeting with different levels of success. Here we outline these efforts in general terms giving just a few examples for each; unfortunately, space does not for allow in-depth reviews, for which we apologise.


Bioorthogonal Dual Functionalization of Self-Assembling Peptide Fibers.

ZN Mahmoud, SB Gunnoo, AR Thomson, JM Fletcher, and DN Woolfson

Biomaterials 32(15) 3712-3720 (2011)

The ability to selectively modify peptide- and protein-based biomaterials under mild conditions and in aqueous buffers is essential to the development of certain areas of bionanotechnology, tissue engineering and synthetic biology. Here we show that Self-Assembling peptide Fibers (SAFs) can incorporate multiple modified peptides non-covalently, stoichiometrically and without disrupting their structure or stability. The modified peptides contain groups suitable for post-assembly click reactions in water, namely azides and alkenes. Labeling of these groups is achieved using the orthogonal Cu(I)-catalyzed azide-alkyne and photoinitiated thiol-ene reactions, respectively. Functionalization is demonstrated through the conjugation of biotin followed by streptavidin-nanogold particles, or rhodamine, and visualized by electron and light microscopy, respectively. This has been shown for fibers harboring either or both of the modified peptides. Furthermore, the amounts of each modified peptide in the fibers can be varied with concomitant changes in decoration. This approach allows the design and assembly of fibers with multiple functional components, paving the way for the development of multi-component functionalized systems.


The Evolution and Structure Prediction of Coiled Coils Across All Genomes.

O Rackham, M Madera, CT Armstrong, TL Vincent, DN Woolfson, and J Gough

J. Mol. Biol. 403, 480-493 (2010)

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available but key issues remain: oligomeric state andprotein-protein interface prediction and extension to all genomes. We present SpiriCoil (, which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds ofhidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils aid. SpiriCoil performs at least as well as existing methods at detecting coiled coils, and significantly advances the state-of-the-art for oligomer-state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (1,200+), and a resulting web interface supplies: data download, alignments, scores, oligomeric state classifications, 3D homology models and visualisation. This has allowed for the first time a genome-wide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life (LUCA). The vast majority of occasions that individual coiled coils have arisen de novo was before LUCA; we do however observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution, and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.


Metal binding to a zinc-finger peptide: a comparison between solution and the gas phase.

Y Berezovskaya, CT Armstrong, AL Boyle, M Porrini, DN Woolfson, and PE Barran

Chem. Commun. 47, 412-414 (2011) DOI: 10.1039/c0cc02445g

Solution-phase spectroscopy and mass spectrometry are used to probe interactions between divalent metal ions and a synthetic Cys2His2 zinc-finger peptide (vCP1). Both methods provide the same order of binding affinity, zinc ≥ cobalt >> copper >> calcium. Collision-cross-section measurements show that both apo and holo forms are compact. This is corroborated by molecular-dynamics simulations.


More than just bare scaffolds: towards multi-component and decorated fibrous biomaterials

DN Woolfson amd ZN Mahmoud

Chem. Soc. Rev. 39, 3464-3479 (2010) DOI: 10.1039/c0cs00032a

We are entering a new phase in biomaterials research in which rational design is being used to produce functionalised materials tailored to specific applications. As is evident from this Themed Issue, there are now a number of distinct types of designed, self-assembling, fibrous biomaterials. Many of these are ripe for development and application for example as scaffolds for 3D cell culture and tissue engineering, and in templating inorganic materials. Whilst a number of groups are making headway towards such applications, there is a general challenge to translate a wealth of excellent basic research into materials with a genuine future in real-life applications. Amongst other contemporary aspects of this evolving research area, a key issue is that of decorating or functionalising what are mostly bare scaffolds. There are a number of hurdles to overcome to achieve effective and controlled labelling of the scaffolds, for instance: maintaining biocompatibility, i.e., by minimising covalent chemistry, or using milder bioconjugation methods; attaining specified levels of decoration, and, in particular, high and stoichiometric labelling; introducing orthogonality, such that two or more functions can be appended to the same scaffold; and, in relevant cases, maintaining the possibility for recombinant peptide/protein production. In this critical review, we present an overview of the different approaches to tackling these challenges largely for self-assembled, peptide-based fibrous systems. We review the field as it stands by placing work within general routes to fibre functionalisation; give worked examples on our own specific system, the SAFs; and explore the potential for future developments in the area. Our feeling is that by tackling the challenges of designing multi-component and functional biomaterials, as a community we stand to learn a great deal about self-assembling biomolecular systems more broadly, as well as, hopefully, delivering new materials that will be truly useful in biotechnology and biomedical applications.


The non-covalent decoration of self-assembling protein fibers.

ZN Mahmoud, DJ Grundy, KJ Channon and DN Woolfson

Biomaterials 31(29), 7468-7474 (2010) DOI: 10.1016/j.biomaterials.2010.06.041

The design of self-assembling fibers presents challenges in basic science, and has potential for developing materials for applications in areas such as tissue engineering. A contemporary issue in the field is the construction of multi-component, functionalized systems. Previously, we have developed peptide-based fibers, the SAF system, that comprises two complementary peptides, which affords considerable control over assembly and morphology. Here we present a straightforward route to functionalizing the SAFs with small molecules and, subsequently, other moieties. This is achieved via non-covalent recruitment of charged peptide tags, which offers advantages such as further control, reversibility, and future prospects for developing recombinant tags. We demonstrate the concept by appending fluorescent labels and biotin (and thence gold nanoparticles) to the peptides, and visualising the resulting decorated SAFs by light and electron microscopy. The peptide tags bind in the nm-mum range, and show specificity compared with control peptides, and for the SAFs over similar alpha-helix-based peptide fibers.


n-->pi* interactions in proteins.

GJ Bartlett, A Choudhary, RT Raines and DN Woolfson

Nat Chem Biol 6(8), 567-568 (2010) DOI: 10.1038/nchembio.406
News and reviews on the topic of this paper can be found at the following links: Review 1 and Review 2

Hydrogen bonds between backbone amides are common in folded proteins. Here, we show that an intimate interaction between backbone amides also arises from the delocalization of a lone pair of electrons (n) from an oxygen atom to the antibonding orbital (pi*) of the subsequent carbonyl group. Natural bond orbital analysis predicted significant n-->pi* interactions in certain regions of the Ramachandran plot. These predictions were validated by a statistical analysis of a large, non-redundant subset of protein structures determined to high resolution. The correlation between these two independent studies is striking. Moreover, the n-->pi* interactions are abundant and especially prevalent in common secondary structures such as alpha-, 3(10)- and polyproline II helices and twisted beta-sheets. In addition to their evident effects on protein structure and stability, n-->pi* interactions could have important roles in protein folding and function, and merit inclusion in computational force fields.


Side-Chain Pairing Preferences in the Parallel Coiled-Coil Dimer Motif: Insight on Ion Pairing between Core and Flanking Sites.

JD Steinkruger, DN Woolfson and SH Gellman

J. Am. Chem. Soc. Communications 132(22), 7586-7588 (2010) DOI: 10.1021/ja100080q

A new strategy for rapid evaluation of sequence−stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol−thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a−a′ side-chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a−g′). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.


Assembly Pathway of a Designed alpha-Helical Protein Fiber.

EHC Bromley, KJ Channon, PJS King, ZN Mahmoud, EF Banwell, MF Butler, MP Crump, TE Dafforn, DMR Hicks, JD Hirst, A Rodger and DN Woolfson

Biophysical Journal 98(8), 1668 - 1676 (2010) DOI: 10.1016/j.bpj.2009.12.4309

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures—for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed α-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.


Building fibrous biomaterials from alpha-helical and collagen-like coiled-coil peptides.

DN Woolfson

Biopolymers: Peptide Science 94(1), 118-127. (2010) DOI: 10.1002/bip.21345

Over the decade and a half, interest has soared in the development of peptide-based biomaterials and their potential applications in biotechnology. This review outlines the advances during this time in the construction of biomaterials based on the alpha-helical coiled-coil and collagen-like peptides. These structures and the resulting designs are distinct from the more-commonly used beta-structured peptides, which often lead to hydrogels comprising amyloid-like fibrils. The review covers basic design rules for these helical assemblies, and the various fibrous biomaterials that can be accomplished with them, which include: rigid structures with straight, branched or networked structures; decorated and functionalised systems, and most-recently flexible fibers and entangled hydrogel networks. This plethora of alpha-helix-based biomaterials, together with more-recent collagen-like assemblies, that are emerging from various laboratories complement those developed using beta-structured peptides, and open exciting new avenues for biomaterials research and potential new application areas.


Modular Design of Peptide Fibrillar Nano- to Microstructures.

MG Ryadnov, A Bella, S Timson, and DN Woolfson

J. Am. Chem. Soc. Communications, 13240-13241 (2009) DOI: 10.1021/ja905539h

A general concept for designing self-assembling peptide fibrillar nano- to microstructures is described. The approach relies on the use of straightforward α-helical one-heptad (1 nm) modules without the need for more-specific designs. Various combinations of the modules gave a variety of constructs that were examined by a combination of spectroscopy and microscopy. These show that simple rearrangements and stereochemical conversions of the modules within similar templates lead to different fiber morphologies. The concept opens new strategies for designing fibrous materials with tunable properties at the nanoscale.


N@d, a coiled-coil motif that sequesters ions to the hydrophobic core.

MD Hartmann, O Ridderbusch, K Zeth, R Albrecht, OD Testa, DN Woolfson, G Sauer, S Dunin-Horkawicz, AN Lupas, and B Hernandez Alvarez

Proc. Natl. Acad. Sci. U. S. A. 16950-16955 (2009) DOI: 10.1073/pnas.0907256106

Most core residues of coiled coils are hydrophobic. Occasional polar residues are thought to lower stability, but impart structural specificity. The coiled coils of trimeric autotransporter adhesins (TAAs) are conspicuous for their large number of polar residues in position d of the core, which often leads to their prediction as natively unstructured regions. The most frequent residue, asparagine (N@d), can occur in runs of up to 19 consecutive heptads, frequently in the motif [I/V]xxNTxx. In the Salmonella TAA, SadA, the core asparagines form rings of interacting residues with the following threonines, grouped around a central anion. This conformation is observed generally in N@d layers from trimeric coiled coils of known structure. Attempts to impose a different register on the motif show that the asparagines orient themselves specifically into the core, even against conflicting information from flanking domains. When engineered into the GCN4 leucine zipper, N@d layers progressively destabilized the structure, but zippers with 3 N@d layers still folded at high concentration. We propose that N@d layers maintain the coiled coils of TAAs in a soluble, export-competent state during autotransport through the outer membrane. More generally, we think that polar motifs that are both periodic and conserved may often reflect special folding requirements, rather than an unstructured state of the mature proteins.


Flow Linear Dichroism of Some Prototypical Proteins.

BM Bulheller, A Rodger, MR Hicks, TR Dafforn, LC Serpell, KE Marshall, EHC Bromley, PJS King, KJ Channon, DN Woolfson, and JD Hirst

J. Am. Chem. Soc. 131, 13305-13314 (2009) DOI: 10.1021/ja902662e

Flow linear dichroism (LD) spectroscopy provides information on the orientation of molecules in solution and hence on the relative orientation of parts of molecules. Long molecules such as fibrous proteins can be aligned in Couette flow cells and characterized using LD. We have measured using Couette flow and calculated from first principles the LD of proteins representing prototypical secondary structure classes: a self-assembling fiber and tropomyosin (all-alpha-helical), FtsZ (an alpha-beta protein), an amyloid fibril (beta-sheet), and collagen [poly(proline)II helices]. The combination of calculation and experiment allows elucidation of the protein orientation in the Couette flow and the orientation of chromophores within the protein fibers.


Rational design of peptide-based building blocks for nanoscience and synthetic biology.

CT Armstrong, AL Boyle, EHC Bromley, ZN Mahmoud, L Smith, AR Thomson, and DN Woolfson

Faraday Discuss. 143, 305-317 (2009) DOI: 10.1039/b901610d

The rational design of peptides that fold to form discrete nanoscale objects, and/or self-assemble into nanostructured materials is an exciting challenge. Such efforts test and extend our understanding of sequence-to-structure relationships in proteins, and potentially provide materials for applications in bionanotechnology. Over the past decade or so, rules for the folding and assembly of one particular protein-structure motif -the alpha-helical coiled coil- have advanced sufficiently to allow the confident design of novel peptides that fold to prescribed structures. Coiled coils are based on interacting alpha-helices, and guide and cement many protein-protein interactions in nature. As such, they present excellent starting points for building complex objects and materials that span the nano-to-micron scales from the bottom up. Along with others, we have translated and extended our understanding of coiled-coil folding and assembly to develop novel peptide-based biomaterials. Herein, we outline briefly the rules for the folding and assembly of coiled-coil motifs, and describe how we have used them in de novo design of discrete nanoscale objects and soft synthetic biomaterials. Moreover, we describe how the approach can be extended to other small, independently folded protein motifs?such as zinc fingers and EF-hands that could be incorporated into more complex, multi-component synthetic systems and new hybrid and responsive biomaterials.


Rational design and application of responsive alpha-helical peptide hydrogels

EF Banwell, ES Abelardo, DJ Adams, MA Birchall, A Corrigan, AM Donald, M Kirkland, LC Serpell, MF Butler, and DN Woolfson

Nat. Mater. 8, 596-600 (2009) DOI: 10.1038/nmat2479

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely alpha-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of alpha-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly . These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.


The Tumbleweed: towards a synthetic protein motor

EHC Bromley, NJ Kuwada, MJ Zuckermann, R Donadini, L Samii, GA Blab, GJ Gemmen, BJ Lopez, PMG Curmi, NR Forde, DN Woolfson, and H Linke

HFSP J. 3, 204-212 (2009) DOI: 10.2976/1.3111282

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function. derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the "Tumbleweed." This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors.


Designed α-Helical Tectons for Constructing Multicomponent Synthetic Biological Systems

EHC Bromley, RB Sessions, AR Thomson, and DN Woolfson

J. Am. Chem. Soc. 131, 928-930 (2009) DOI: 10.1021/ja804231a

One possible route to develop new synthetic-biological systems is to assemble discrete nanoscale objects from programmed peptide-based building blocks. We describe an algorithm to design such blocks based on the coiled-coil protein-folding motif. The success of the algorithm is demonstrated by the production of six peptides that form three target parallel, blunted-ended heterodimers in preference to any of the other promiscuous pairings and alternate configurations, for example, homodimers, sticky-ended assemblies, and antiparallel arrangements. The peptides were linked to promote the assembly of larger, defined nanoscale rods, thus demonstrating that targeted peptide-peptide interactions can be specified in complex mixtures.


A Periodic Table of Coiled-Coil Protein Structures

E Moutevelis and DN Woolfson

J. Mol. Biol 385, 726-732 (2009)

Coiled coils are protein structure domains with two or more α-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a “periodic table of coiled coils,” which we have made available at In this table, coiled-coil assemblies are arranged in columns with increasing numbers of α-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.


CC+: a relational database of coiled-coil structures

OD Testa, E Moutevelis, and DN Woolfson

Nucleic Acids Res. 37, D315-D322 (2009)

We introduce the CC+ Database, a detailed, searchable repository of coiled-coil assignments, which is freely available at Coiled coils were identified using the program SOCKET, which locates coiled coils based on knobs-into-holes packing of side chains between α-helices. A method for determining the overall sequence identity of coiled-coil sequences was introduced to reduce statistical bias inherent in coiled-coil data sets. There are two points of entry into the CC+ Database: the “Periodic Table of Coiled-coil Structures”, which presents a graphical path through coiled-coil space based on manually validated data, and the “Dynamic Interface”, which allows queries of the database at different levels of complexity and detail. The latter entry level, which is the focus of this article, enables the efficient and rapid compilation of subsets of coiled-coil structures. These can be created and interrogated with increasingly sophisticated pull-down, keyword and sequence-based searches to return detailed structural and sequence information. Also provided are means for outputting the retrieved coiled-coil data in various formats, including PyMOL and RasMol scripts, and Position-Specific Scoring Matrices (or amino-acid profiles), which may be used, for example, in protein-structure prediction.


MagicWand: A Single, Designed Peptide That Assembles to Stable, Ordered α-Helical Fibers.

C Gribbon, KJ Channon, WJ Zhang, EF Banwell, EHC Bromley, JB Chaudhuri, ROC Oreffo and DN Woolfson

Biochemistry 47, 10365-10371 (2008)

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric α-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg, with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesis that is, as opposed to blunt-ended discrete species the terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled α-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positions combined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.


Templating Silica Nanostructures on Rationally Designed Self-Assembled Peptide Fibers

SC Holmstrom, PJS King, MG Ryadnov, MF Butler, S Mann, and DN Woolfson

Langmuir 24, 11778-11783 (2008)

Nature presents exquisite examples of templating hard, functional inorganic materials on soft, self-assembled organic substrates. An ability to mimic and control similar processes in the laboratory would increase our understanding of fundamental science, and may lead to potential applications in the broad arena of bionanotechnology. Here we describe how self-assembled, α-helix-based peptide fibers of de novo design can promote and direct the deposition of silica from silicic acid solutions. The peptide substrate can be removed readily through proteolysis, or other facile means to render silica nanotubes. Furthermore, the resulting silica structures, which span the nanometer to micrometer range, can themselves be used to template the deposition of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride). Finally, the peptide-based substrates can be engineered prior to silicification to alter the morphology and mechanical properties of the resulting hybrid and tubular materials.


Synthetic biology through biomolecular design and engineering

K Channon, EHC Bromley, and DN Woolfson

Curr. Opin. Struct. Biol. 18, 491-498 (2008)

Synthetic biology is a rapidly growing field that has emerged in a global, multidisciplinary effort among biologists, chemists, engineers, physicists, and mathematicians. Broadly, the field has two complementary goals: To improve understanding of biological systems through mimicry and to produce bioorthogonal systems with new functions. Here we review the area specifically with reference to the concept of synthetic biology space, that is, a hierarchy of components for, and approaches to generating new synthetic and functional systems to test, advance, and apply our understanding of biological systems. In keeping with this issue of Current Opinion in Structural Biology, we focus largely on the design and engineering of biomolecule-based components and systems.


Electrostatic Control of Thickness and Stiffness in a Designed Protein Fiber

D Papapostolou, EHC Bromley, C Bano, and DN Woolfson

J. Am. Chem. Soc. 130, 5124-5130 (2008)

Attempts to design peptide-based fibers from first principles test our understanding of protein folding and assembly, and potentially provide routes to new biomaterials. Several groups have presented such designs based on α-helical and β-strand building blocks. A key issue is this area now is engineering and controlling fiber morphology and related properties. Previously, we have reported the design and characterization of a self-assembling peptide fiber (SAF) system based on alpha-helical coiled-coil building blocks. With preceding designs, the SAFs are thickened, highly ordered structures in which many coiled coils are tightly bundled. As a result, the fibers behave as rigid rods. Here we report successful attempts to design new fibers that are thinner and more flexible by further programming at the amino-acid sequence level. This was done by introducing extended, or "smeared", electrostatic networks of arginine and glutamate residues to the surfaces of the coiled-coil building blocks. Furthermore, using arginine rather than lysine in these networks plays a major role in the fiber assembly, presumably by facilitating multidentate intra and intercoiled-coil salt bridges.


Peptide and Protein Building Blocks for Synthetic Biology: From Programming Biomolecules to Self-Organized Biomolecular Systems

EHC Bromley, K Channon, E Moutevelis, and DN Woolfson

ACS Chem. Biol. 3, 38-50 (2008)

There are several approaches to creating synthetic-biological systems. Here, we describe a molecular-design approach. First, we lay out a possible synthetic-biology space, which we define with a plot of complexity of components versus divergence from nature. In this scheme, there are basic units, which range from natural amino acids to totally synthetic small molecules. These are linked together to form programmable tectons, for example, amphipathic α-helices. In turn, tectons can interact to give self-assembled units, which can combine and organize further to produce functional assemblies and systems. To illustrate one path through this vast landscape, we focus on protein engineering and design. We describe how, for certain protein-folding motifs, polypeptide chains can be instructed to fold. These folds can be combined to give structured complexes, and function can be incorporated through computational design. Finally, we describe how protein-based systems may be encapsulated to control and investigate their functions.


Preferred side-chain constellations at antiparallel coiled-coil interfaces

EB Hadley, OD Testa, DN Woolfson, and SH Gellman

Proc. Natl. Acad. Sci. U. S. A. 105, 530-535 (2008)

Reliable predictive rules that relate protein sequence to structure would facilitate postgenome predictive biology and the engineering and de novo design of peptides and proteins. Through a combination of experiment and analysis of the protein data bank (PDB), we have deciphered and rationalized new rules for helix-helix interfaces of a common protein-folding and association motif, the antiparallel dimeric coiled coil. These interfaces are defined by a specific pattern of interactions among largely hydrophobic side chains often referred to as knobs-into-holes (KIH) packing: a knob from one helix inserts into a hole formed by four residues on the partner. Previous work has focused on lateral interactions within the KIH motif, for example, between an a position on one helix and a d' position on the other in an antiparallel coiled coil. We show that vertical interactions within the KIH motif, such as a'-a-a', are energetically important as well. The experimental and database analyses concur regarding preferred vertical combinations, which can be rationalized as leading to favorable side-chain interactions that we call constellations. The findings presented here highlight an unanticipated level of complexity in coiled-coil interactions, and our analysis of a few specific constellations illustrates a general, multipronged approach to addressing this complexity.


Self-Assembled Templates for Polypeptide Synthesis

MG Ryadnov and DN Woolfson

J. Am. Chem. Soc. 129, 14074-14081 (2007)

The chemical synthesis of polypeptide chains >50 amino acids with prescribed sequences is challenging. In one approach, native chemical ligation (NCL), short, unprotected peptides are connected through peptide bonds to render proteins in water. Here we combine chemical ligation with peptide self-assembly to deliver extremely long polypeptide chains with stipulated, repeated sequences. We use a self-assembling fiber (SAF) system to form structures tens of micrometers long. In these assemblies, tens of thousands of peptides align with their N- and C-termini abutting. This arrangement facilitates chemical ligation without the usual requirement for a catalytic cysteine residue at the reactive N-terminus. We introduced peptides with C-terminal thioester moieties into the SAFs. Subsequent ligation and disassembly of the noncovalent components produced extended chains 10µm long and estimated at ≥3 MDa in mass. These extremely long molecules were characterized by a combination of biophysical, hydrodynamic, and microscopic measurements.


Atypical bZIP domain of viral transcription factor contributes to stability of dimer formation and transcriptional function

C Schelcher, S Al Mehairi, E Verrall, Q Hope, K Flower, B Bromley, DN Woolfson, MJ West, and AJ Sinclair

J. Virol. 81, 7149-7155 (2007)

We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded α-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as α-helices as designed. These patterns extend unbroken across the widths (50 nm) and lengths (>10 μm) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptide-design process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.


Engineering nanoscale order into a designed protein fiber

D Papapostolou, AM Smith, EDT Atkins, SJ Oliver, MG Ryadnov, LC Serpell, and DN Woolfson

Proc. Natl. Acad. Sci. U. S. A. 104, 10853-10858 (2007)

We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded α-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as α-helices as designed. These patterns extend unbroken across the widths (50 nm) and lengths (>10 μm) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptide-design process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.


Protein-small molecule interactions in neocarzinostatin, the prototypical enediyne chromoprotein antibiotic

JR Baker, DN Woolfson, FW Muskett, RG Stoneman, MD Urbaniak, and S Caddick

Chembiochem 8, 704-717 (2007)

The enediyne chromoproteins are a class of potent antitumour antibiotics comprising a 1:1 complex of a protein and a noncovalently bound chromophore. The protein is required to protect and transport the highly labile chromophore, which acts as the cytotoxic component by reacting with DNA leading to strand cleavage. A derivative of the best-studied member of this class, neocarzinostatin (NCS), is currently in use as a chemotherapeutic in Japan. The application of the chromoproteins as therapeutics along with their unique mode of action has prompted widespread interest in this area. Notable developments include the discovery of non-natural ligands for the apoproteins and the observation that multiple binding modes are available for these ligands in the binding site. Mutation studies on the apoproteins have revealed much about their stability and variability, and the application of an in vitro evolution method has conferred new binding specificity for unrelated ligands. These investigations hold great promise for the application of the apoproteins for drug-delivery, transport and stabilisation systems.


Kinking the Coiled Coil – Negatively Charged Residues at the Coiled-coil Interface

R Straussman, A Ben-Ya'acov, DN Woolfson, and S Ravid

J. Mol. Biol. 366, 1232-1242 (2007)

The coiled coil is one of the most common protein-structure motifs. It is believed to be adopted by 3–5% of all amino acids in proteins. It comprises two or more α-helical chains wrapped around one another. The sequences of most coiled coils are characterized by a seven-residue (heptad) repeat, denoted (abcdefg)n. Residues at the a and d positions define the helical interface (core) and are usually hydrophobic, though about 20% are polar or charged. We show that parallel coiled-coils have a unique pattern of their negatively charged residues at the core positions: aspartic acid is excluded from these positions while glutamic acid is not. In contrast the antiparallel structures are more permissive in their amino acid usage. We show further, and for the first time, that incorporation of Asp but not Glu into the a positions of a parallel coiled coil creates a flexible hinge and that the maximal hinge angle is being directly related to the number of incorporated mutations. These new computational and experimental observations will be of use in improving protein-structure predictions, and as rules to guide rational design of novel coiled-coil motifs and coiled coil-based materials.


Microwave enhanced palladium catalysed coupling reactions: A diversity-oriented synthesis approach to functionalised flavones

RJ Fitzmaurice, ZC Etheridge, E Jumel, DN Woolfson, and S Caddick

Chem. Commun. 46, 4814-4816 (2006)

Microwave enhanced diversity-oriented synthesis (MEDOS) using palladium catalysed protocols is introduced as a powerful new strategy for the synthesis of systematically modified small molecules and is highlighted by application to functionalised flavones.


Peptide-based fibrous biomaterials: some things old, new and borrowed.

DN Woolfson and MG Ryadnov

Curr. Opin. Chem. Biol. 10, 559-567 (2006)

Bioinspired fibrous materials that span the nano-to-meso scales have potentially broad applications in nanobiotechnology; for instance, as scaffolds in 3D cell culture and tissue engineering, and as templates for the assembly of other polymer and inorganic materials. The field is burgeoning, and this review is necessarily focused. It centres on recent developments in the design of peptide-based fibres and particularly those using the alpha-helix and the collagen triple helix as building blocks for self-assembly. Advances include new designs in both categories, the assembly of more-complex topologies using fibres themselves as building blocks, and the decoration of the assembled materials with functional moieties.


Engineering Increased Stability into Self-Assembled Protein Fibers.

AM Smith, EF Banwell, WR Edwards, MJ Pandya, and DN Woolfson

Adv. Funct. Mater. 16, 1022-30 (2006) DOI: 10.1002/adfm.200500568

Two stages in the rational redesign of a peptide-based, self-assembling fiber (SAF) are described. The SAF system comprises two peptides designed to form an offset a-helical coiled-coil heterodimer. The 'sticky-ends' are complementary and promote longitudinal assembly. Alone, the two peptides are unstructured, but co-assemble upon mixing to form a-helical fibrils, which bundle to form fibers 40-50 nm wide and tens of micrometers long. Assembly is controllable and occurs at pH 7 in water, making SAFs a potential scaffold for 3D cell culture. The purposes of the redesigns were 1) to investigate the fiber-thickening process, and 2) to increase fiber stability for potential biological and biomedical applications. First, mutations were made to the original peptide designs to increase fibril-fibril interactions and so produce thicker and more-stable fibers. The second iteration aimed to increase the primary peptide-peptide interactions by increasing the overlap in the offset dimer and so promote the initial step in fiber formation. As judged by circular dichroism spectroscopy and transmission electron microscopy, both iterations improved fiber assembly and stability: the critical peptide concentration for assembly improved from 60 uM to 4 uM the midpoint of thermal unfolding increased from 22 degrees C to 65 degrees C; and the salt tolerance improved from 75 mM to greater than 250 mM KCl. These improvements bring closer applications of the SAF system under physiological conditions, for example as a biocompatible material for 3D cell culture. In addition, ordered surface features were observed in the second- and third-generation fibers compared with the original design. This indicates improved internal order in the redesigned fibers. In turn, this suggests a molecular mechanism for the improved stability and sheds light on the fiber-assembly process.


Synthetic ligands for apo-neocarzinostatin.

S Caddick, FW Muskett, RG Stoneman, and DN Woolfson

J. Am. Chem. Soc 128, 4204-5 (2006)

Neocarzinostatin (NCS) is a 1:1 complex of an enediyne chromophore (NCSChrom), non-covalently bound to an 11 kDa protein (apoNCS). We are exploring apoNCS as a generic protein system for sequestering small molecules for therapeutic applications. Here, we disclose a new flavone ligand 1 for apoNCS and present a high-resolution NMR structure of this ligand bound to apoNCS. This is the first high-resolution structure of a completely non-cognate ligand bound to the apoNCS protein. This work provides unambiguous evidence that a completely new class of ligand can bind specifically to apoNCS. Furthermore, the mode of binding is different than that of the naphthoate-based ligands, and for such a simple hydrophobic compound, the new ligand surprisingly binds specifically. This work indicates that apo-Neocarzinostatin has multiple selective and distinct binding modes for small-molecule cargo.


Amino acid pairing preferences in parallel beta-sheets in proteins.

HM Fooks, ACR Martin, DN Woolfson, RB Sessions, and EG Hutchinson

J. Mol. Biol. 356, 32-44 (2006)

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (


ZiCo: A Peptide Designed to Switch Folded State upon Binding Zinc.

E Cerasoli, BK Sharpe, and DN Woolfson

J. Am. Chem. Soc 127, 15008-9 (2005)

We describe a novel approach to the design of a metal-triggered conformational switch. Specifically, two distinct protein-folding motifs were merged into one polypeptide sequence. The target structures were an alpha-helical coiled-coil trimer and zinc-bound monomer. Solution-phase spectroscopic, sedimentation, and binding studies confirmed the key aspects of the design. Both forms of the peptide were cooperatively folded, and the switch between them was reversible. This design process potentially presents a novel route to peptide-based biosensors.


The design of coiled-coil structures and assemblies

DN Woolfson

Adv. Prot. Chem. 70, 79-112 (2005)

Protein design allows sequence-to-structure relationships in proteins to be examined and, potentially, new protein structures and functions to be made to order. To succeed, however, the protein-design process requires reliable rules that link protein sequence to structure?function. Although our present understanding of coiled-coil folding and assembly is not complete, through numerous bioinformatics and experimental studies there are now sufficient rules to allow confident design attempts of naturally observed and even novel coiled-coil motifs. This review summarizes the current design rules for coiled coils, and describes some of the key successful coiled-coil designs that have been created to date. The designs range from those for relatively straightforward, naturally observed structures-including parallel and antiparallel dimers, trimers and tetramers, all of which have been made as homomers and heteromers-to more exotic structures that expand the repertoire of Nature's coiled-coil structures. Examples in the second bracket include a probe that binds a cancer-associated coiled-coil protein; a tetramer with a right-handed supercoil; sticky-ended coiled coils that self-assemble to form fibers; coiled coils that switch conformational state; a three-component two-stranded coiled coil; and an antiparallel dimer that directs fragment complementation of larger proteins. Some of the more recent examples show an important development in the field; namely, new designs are being created with function as well as structure in mind. This will remain one of the key challenges in coiled-coil design in the next few years. Other challenges that lie ahead include the need to discover more rules for coiled-coil prediction and design, and to implement these in prediction and design algorithms. The considerable success of coiled-coil design so far bodes well for this, however. It is likely that these challenges will be met and surpassed.


MaP peptides: Programming the self-assembly of peptide-based mesoscopic matrices

MG Ryadnov and DN Woolfson

J. Am. Chem. Soc 127, 12407-12415 (2005)

We describe an approach that utilizes nonlinear peptides to direct the assembly of previously reported Self-Assembling Fibers (SAFs). The SAF system comprises two complementary linear peptides, SAF-p1 and SAF-p2a, which combine to form exclusively linear, nonbranched fibers. The Matrix-Programming (MaP) peptides described herein are based on these peptides: they comprise two or three half-peptide blocks derived from the SAF peptides, which are conjugated via dendritic hubs. Different MaP peptides coassembled with the standard SAF peptides to form specific structures, such as hyperbranched networks, polygonal matrices, and regularly segmented and terminated fibers. The role of each half-peptide block in dictating the different features has been elucidated. This provides a strong basis for designing new peptide-based nanostructured materials from the bottom up.


Polar assembly in a designed protein fiber

AM Smith, SFA Acquah, N Bone, HW Kroto, MG Ryadnov, MSP Stevens, DRM Walton, and DN Woolfson

Angew. Chem. Int. Ed. 44, 325-328 (2005) DOI: 10.1002/anie.200461599

This publication has no abstract.


Sequence and Structural Duality: Designing Peptides to Adopt Two Stable Conformations

MJ Pandya, E Cerasoli, A Joseph, RG Stoneman, E Waite, and DN Woolfson

J. Am. Chem. Soc 126, 17016-17024 (2004) DOI: 10.1021/ja045568c

Abstract: To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intra-molecularly disulfide bridged state. Using an iterative process we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.


Design and synthesis of a nitrogen-mustard derivative stabilised by apo-Neocarzinostatin

MD Urbaniak, JP Bingham, JA Hartley, DN Woolfson, and S Caddick

J. Med. Chem. 47, 4710-4715 (2004) DOI: 10.1021/jm040790d

Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.


Biophysical and mutational analysis of the putative bZIP domain of Epstein-Barr virus EBNA 3C.

MJ West, HM Webb, AJ Sinclair, and DN Woolfson

J. Virol. 78, 9431-9445 (2004) DOI: 10.1128/JVI.78.17.9431-9445.2004

Epstein-Barr virus nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization and functions as a regulator of viral and cellular transcription. EBNA 3C contains glutamine-rich and proline-rich domains and a region in the N terminus consisting of a stretch of basic residues followed by a run of leucine residues spaced seven amino acids apart. This N-terminal domain is widely believed to represent a leucine zipper dimerization motif (bZIP). We have performed the first structural and functional analysis of this motif and demonstrated that this domain is not capable of forming stable homodimers. Peptides encompassing the EBNA 3C zipper domain are approximately 54 to 67% alpha-helical in solution but cannot form dimers at physiologically relevant concentrations. Moreover, the EBNA 3C leucine zipper cannot functionally substitute for another homodimerizing zipper domain in domain-swapping experiments. Our data indicate, however, that the EBNA 3C zipper domain behaves as an atypical bZIP domain and is capable of self-associating to form higher-order alpha-helical oligomers. Using directed mutagenesis, we also identified a new role for the bZIP domain in maintaining the interaction between EBNA 3C and RBP-JK in vivo. Disruption of the helical nature of the zipper domain by the introduction of proline residues reduces the ability of EBNA 3C to inhibit EBNA 2 activation and interact with RBP-JK in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.


Fiber Recruiting (FiRe) Peptides: Non-Covalent Decoration of an Engineered Protein Scaffold

MG Ryadnov and DN Woolfson

J. Am. Chem. Soc. 126, 7454-7455 (2004) DOI: 10.1021/ja048144r

Fiber Recruiting (FiRe) peptides are described. These are derivatives of self-assembling fiber (SAF) forming peptides that are conjugated with small molecules (in our case, biotin or a FLAG-tag octapeptide). The FiRe peptides are co-assembled into fibers and used as bait to recruit folded and functional proteins to the fiber surfaces. This was demonstrated using two molecular recognition models: namely, a protein-ligand interaction (biotin-streptavidin) and an antigen-antibody (FLAG octapeptide-anti-FLAG-antibody) interaction. This concept offers an approach to mimicking in natural fibrillar systems, such as collagen or fibrin, that communicate specifically with their environments via incorporated or decorated active peptide and protein components.


Engineered and Designed Peptide-based Fibrous Biomaterials

CE MacPhee and DN Woolfson

Curr. Opin. Solid State Mat. Sci. 8, 141-149 (2004) DOI: 10.1016/j.cossms.2004.01.010

Our increasing understanding of peptide and protein folding and assembly raises new possibilities for engineering novel self-assembling supramolecular structures and bio-inspired materials. Here we focus on peptides designed de novo, and natural systems that have been engineered to form extended protein fibres. Potential applications of such assemblies include the preparation of functionalised biomaterials for the development of new diagnostic devices, scaffolds to recruit cells for cell/tissue engineering, and templates for the assembly of inorganic materials.


Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin

CG Benitez-Cardoza, K Stott, M Hirshberg, HM Went, DN Woolfson, and SE Jackson

Biochemistry 43, 5195-5203 (2004) DOI: 10.1021/bi0361620

The stability, dynamic, and structural properties of ubiquitin and two Multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I3F, L15V. V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well- folded and show similar backbone dynamics (T-1, T-2, and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R-factor and R-free are 0.198 and 0.248. respectively, at 2.18 Angstrom resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably. there are almost no changes in the positions of the C-alpha atoms along the entire backbone, and the hydrogen- bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.


Extended knobs-into-holes packing in classical and complex coiled-coil assemblies

J Walshaw and DN Woolfson

J. Struct. Biol 144, 349-361 (2003) DOI: doi:10.1016/j.jsb.2003.10.014

This year marks the 50th anniversary of Crick's seminal paper on the packing of a-helices into coiled-coil structures. The central tenet of Crick's work is the interdigitation of side chains, which directs the helix-helix interactions; so called knobs-into-holes packing. Subsequent determinations of coiled-coil-protein sequences and structures confirmed the key features of Crick's model and established it as a fundamental concept in structural biology. Recently, we developed a program, SOCKET, to recognise knobs-into-holes packing in protein structures, which we applied to the Protein Data Bank to compile a database of coiled-coil structures. In addition to classic structures, the database reveals 4-helix bundles and larger helical assemblies. Here, we describe how the more-complex structures can be understood by extending Crick's principles for classic coiled coils. In the simplest case, each helix of a 2-stranded structure contributes a single seam of (core) knobs-into-holes to the helical interface. 3-, 4-, and 5-Stranded structures, however, are best considered as rings of helices with cycles of knobs-into-holes. These higher-order oligomers make additional (peripheral) knobs-into-holes that broaden the helical contacts. Combinations of core and peripheral knobs may be assigned to different sequence repeats offset within the same helix. Such multiple repeats lead to multi-faceted helices, which explain structures above dimers. For instance, coiled-coil oligomer state correlates with the offset of the different repeats along a sequence. In addition, certain multi-helix assemblies can be considered as conjoined coiled coils in which multi-faceted helices participate in more than one coiled-coil motif.


"Belt and Braces": A Peptide-Based Linker System of de Novo Design

MG Ryadnov, B Ceyhan, CM Niemeyer, and DN Woolfson

J. Am. Chem. Soc. 125, 9388-9394 (2003) DOI: 10.1021/ja0352045

A new self-assembling peptide-based linker is described. The system comprises three leucine-zipper sequences of de novo design: one peptide, "the belt", templates the co-assembly of the other two half-sized peptides, "the braces". These basic features were confirmed by circular dichroism spectroscopy and analytical ultracentrifugation: when mixed the three peptides reversibly formed a predominantly helical and stable 1:1:1 ternary complex. Surface plasmon resonance experiments demonstrated assembly of the complex on gold surfaces, whilst the ability of the system to bring together peptide-bound cargo was demonstrated using colloidal gold nanoparticles. In the latter experiments, the nanoparticles were derivatized with the brace peptides prior to the addition of the belt. Transmission electron microscopy images of the resulting networks revealed regular 7 nm separations between adjacent particles, consistent with the 42-amino acid helical design of the belt and braces. To our knowledge, belt and braces is a novel concept in leucine-zipper assembly, and the first example of employing peptides to guide nanoparticle assembly.


Introducing Branches into a Self-Assembling Peptide Fiber

MG Ryadnov and DN Woolfson

Angew. Chem.-Int. Edit. 42, 3021-3023 (2003) DOI: 10.1002/anie.200351418

There is a growing interest in employing peptides as building blocks in the self-assembly of supramolecular structures.[1] Such assemblies have potential as novel scaffolds for functionalizing surfaces and in tissue engineering.[2] Elsewhere,[3] we describe a self-assembling fiber (SAF) system comprising two designed peptides (1 & 2, aka SAF-p1 and SAF-2a, Figure 1). These sequences are based on established design principles for leucine-zipper motifs.[4] However, unlike all other leucine zippers, which are blunt ended, the SAF peptides are designed to assemble with sticky ends that facilitate fibrillogenesis (Figure 1, Figure S1). Like other systems,[1a,c,f] the SAF peptides are linear and form exclusively linear and non-branching fibers when mixed; n.b. 2 is a slight redesign of the previously described SAF-p2,[3] this new design combines with 1 to give more-stable and better-ordered linear fibers (Figure S2A-D, supporting information). Natural protein fibers such as those formed by actin, collagen and fibrin branch. We set out to design special units to complement the standard SAF building blocks and, so, engineer branched fibers de novo. Here we report T-shaped peptides, T-SAFs, that co-assemble with the standard SAF peptides to give branched self-assembling fibers. In T-SAFs the ?bar? of the ?T? is the complete SAF peptide 2 and the ?stem? (peptide 3) is the N-terminal half of 2; the stem and the bar are joined via a linker, typically of three b-alanine (bAla) units, between the C-terminus of peptide 3 and the e-amino group of the central lysine (lysine-14) of peptide 2 (Figure 1). In principle, when combined with peptides 1 and 2, T-SAFs should promote assembly of orthogonally conjoined fibers.


Engineering the morphology of a self-assembling protein fibre

MG Ryadnov and DN Woolfson

Nat. Mater. 2, 329-332 (2003) DOI: 10.1038/nmat885

Biological assemblies provide inspiration for the development of new materials for a variety of applications. Our ability to realize this potential, however, is hampered by difficulties in producing and engineering natural biomaterials, and in designing them from new. We previously described a self-assembling system comprising two short complementary segments of straight synthetic polypeptides (dubbed standards in this report). Their interaction results in the formation of long fibres ? about 50 nm in diameter ? that extend straight and without branching for tens to hundreds of micrometres. Our aim is to influence and, ultimately, to control fibre morphology. Here, we show that the standard peptides can be supplemented with special peptides to effect morphological changes in the fibres. Specifically, we created half-sized subunits of the standard peptides, which were combined to make nonlinear peptides. When mixed with the standard peptides, these nonlinear peptides produced kinked, waved and branched fibres. We related the numbers of these features to the special/standard ratios empirically. Furthermore, the extent and frequency of kinking was altered by changing the standard-fibre background: more kinking was observed in a background of thinner, less-stable fibres. The ability to perform such transformations holds promise for bottom-up assembly and engineering-responsive mimetic materials for applications in surface and tissue engineering.


Chemical Synthesis and Cytotoxicity of Dihydroxylated Cyclopentenone Analogues of Neocarzinostatin Chromophore

MD Urbaniak, LM Frost, JP Bingham, LR Kelland, JA Hartley, DN Woolfson, and S Caddick

Bioorg. Med. Chem. Lett. 13, 2025-2027 (2003) DOI: 10.1016/S0960-894X(03)00328-7

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.


Solution Structure of a Novel Chromoprotein Derived from Apo-Neocarzinostatin and a Synthetic Chromophore.

MD Urbaniak, FW Muskett, MD Finucane, S Caddick, and DN Woolfson

Biochemistry 41, 11731-11739 (2002) DOI: 10.1021/bi0262146

The natural complex Neocarzinostatin comprises a labile chromophore non-covalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apo-protein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a similar location to the natural chromophore, but with a distinct orientation. These results provide structural evidence that the apo-protein can readily accommodate small drug-like entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex to act as a drug-delivery system described by others, together with the promiscuity in binding reported here, suggests potential applications for small-molecule binding by apo-Neocarzinostatin.


Mini-proteins Trp the light fantastic

SH Gellman and DN Woolfson

Nature Struct. Biol. 9, 6, 408-410 (2002) DOI: 10.1038/nsb0602-408

A new 20-residue peptide represents the smallest example to date of cooperatively folded tertiary structure. This achievement provides a new tool for elucidating protein conformational preferences. The mini-protein should serve as a fruitful platform for protein design.


Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

G Siligardi, B Panaretou, P Meyer, S Singh, DN Woolfson, PW Piper, LH Pearl, and C Prodromou

J. Biol. Chem. 277, 20151-20159 (2002)

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle, and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a 'scaffold', recruiting Hsp70 and the bound client to Hsp90 early in the cycle, and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialised co-chaperone, Cdc37p/p50cdc37 whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50cdc37 like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a 'relaxed' ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50cdc37 forms a stable complex with geldanamycin-bound Hsp90, and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.


Generalised Crick equations for modelling non-canonical coiled coils

G Offer, MR Hicks, and DN Woolfson

J. Struct. Biol. 137, 41-53 (2002)

Crick envisaged the a-helical coiled coil to result from systematic bending of an a-helix such that every seventh residue was structurally equivalent and derived equations for the coordinates of the backbone atoms. Crick¹s predictions were vindicated experimentally and coiled-coil sequences shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs. The supercoiling of such coiled coils varies with the sequence pattern, being left or right-handed in purely heptad-based or hendecad-based motifs, respectively. To model such structures, we describe how the Crick equations can be extended to coiled coils with a mixture of motifs where the pitch is not constant. Using the analogy of the bending of a beam, we took the tilt angle to change linearly along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the a-helical strands. We tested our approach by fitting the two, three and four-stranded non-canonical coiled coils of GrpE, haemagglutinhemagglutinin and tetrabrachion. The backbone atoms of the model and crystal structures agreed with rms deviations of <1.1 Angstroms.


Investigating the tolerance of coiled-coil peptides to non-heptad sequence inserts

MR Hicks, J Walshaw, and DN Woolfson

J. Struct. Biol. 137, 73-81 (2002)

Coiled-coil motifs foster a wide variety of protein-protein interactions. Canonical coiled coils are based on seven-residue repeats, which guide the folding and assembly of amphipathic a-helices. In many cases such repeats remain unbroken for tens to hundreds of residues. However, the sequences of an increasing number of putative and characterised coiled coils digress from this pattern. We probed the consequences of non-heptad inserts using a designed leucine-zipper system. The parent peptide, SKIP0, which had four contiguous heptads, was confirmed as a parallel homodimer by circular dichroism spectroscopy and analytical ultracentrifugation. Seven daughter peptides were constructed in which one to seven alanine residues were inserted between the central heptads of SKIP0. Like SKIP0, SKIP7 formed a stable helical dimer, but the other peptides were highly destabilised, with the order of dimer stability SKIP4 >> SKIP5 > SKIP6 > SKIP3 > SKIP2 > SKIP1. These results are consistent with an extended theory of coiled-coil assembly in which coiled-coil-compatible motifs are based on three- and four-residue spacings, and most notably heptad (seven-residue) and hendecad (eleven-residue) repeats. Thus, they help explain why in natural sequences, inserts after canonical heptad repeats most commonly of four-residues. Possible biological roles for non-heptad inserts are discussed.


A designed system for assessing how sequence affects alpha-to-beta conformational transitions in proteins

B Ciani, EG Hutchinson, RB Sessions, and DN Woolfson

J. Biol. Chem. 277, 10150-10155 (2002)

The role of amino-acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study a-to-b conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an a-helical leucine zipper and a b-hairpin. The parent peptide, Template-a, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce b-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-aT. Unlike the parent peptide, Template-aT underwent a heat-inducible switch to b-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilisation of the native states facilitate transitions to amyloid, destabilisation of the leucine-zipper form of Template-aT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative b-hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template-a, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favour b-structure promote amyloid formation.


Core-directed Protein Design

DN Woolfson

Curr. Opin. Struct. Biol. 11, 464-471 (2001)

For various reasons, it seems sensible to redesign or design proteins from the inside out. Past approaches in this field have involved iterations of mutagenesis and characterisation to 'evolve' designs. Increasingly, combinatorial approaches are being taken to select 'fit' sequences from libraries of variant proteins. In particular, in silico methods have been used to good effect. More recently, experimental methods have been developed and improved. We are now in a position to redesign stability and function into natural protein frameworks confidently and to attempt de novo designs for more ambitious targets.


Guidelines For the Assembly of Novel Coiled-coil Structures: Alpha-sheets and Alpha-cylinders.

J Walshaw, JM Shipway, and DN Woolfson

Biochem. Soc. Symposium 68: From Protein Folding to New Enzymes 111-123 (2001)

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a 7-residue (heptad) sequence repeat. With positions of this repeat labelled a-b-c-d-e-f-g residues at a and d tend to be hydrophobic. Such sequences form amphipathic alpha-helices, which assemble into helical bundles via knobs-into-holes (KIH) interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank (PDB). Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues; i.e. a/f'-b/g'-c/a'-d/b'-e/c'-f/d'-g/e'. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open "alpha-sheets" and closed "alpha-cylinders". We highlight these with descriptions of natural structures and outline possibilities for protein design.


Biophysical Analysis of Natural Variants of the Multimerization Region of Epstein-Barr Virus Lytic-switch Protein Bzlf1

MR Hicks, S Balesaria, C Medina-Palazon, MJ Pandya, DN Woolfson, and AJ Sinclair

J. Virol. 75, 5381-5384 (2001)

BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV) replication. On the basis of limited sequence homology and mutagenesis experiments, BZLF1 has been described as a member of the bZip family of transcription factors, but this prospect has not been rigorously tested to date. Here, we present biophysical analysis of the multimerization domain of BZLF1, from three natural variants of EBV, and demonstrate for the first time that the region between amino acids 196 and 227 is sufficient to direct folding as a coiled-coil dimer in vitro.


Socket: a Program For Identifying and Analysing Coiled-coil Motifs Within Protein Structures

J Walshaw and DN Woolfson

J. Mol. Biol. 307, 1427-1450 (2001) DOI: 10.1006/jmbi.2001.4545

The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions. Coiled coils comprise two or more a-helices that wind around each other to form supercoils. The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat. Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states. To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures. We used SOCKET to gather a set of unambiguous coiled-coil structures from the Protein Data Bank (PDB). Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains. SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers. Structures retrieved from the PDB included parallel and anti-parallel variants of 2-, 3- and 4-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled-coil. We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design. To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino-acid profiles for the heptad repeats of different motifs.


Open-and-shut Cases in Coiled-coil Assembly: Alpha-sheets and Alpha-cylinders

J Walshaw and DN Woolfson

Protein Sci 10, 668-673 (2001) DOI: 10.1110/ps.36901

The coiled coil is a ubiquitous protein-folding motif. It is generally accepted that coiled coils are characterised by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic alpha-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed knobs-into-holes packing. We developed software, SOCKET, to recognise this packing in protein structures. As expected, in a trawl of the protein data bank we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, however, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by 150-160 degrees around the alpha-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multi-helix assemblies. Accordingly, we refer to these two forms as alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.


Sticky-end Assembly of a Designed Peptide Fiber Provides Insight Into Protein Fibrillogenesis

MJ Pandya, GM Spooner, M Sunde, JR Thorpe, A Rodger, and DN Woolfson

Biochemistry 39, 8728-8734 (2000) DOI: 10.1021/bi000246g

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. Furthermore, X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred microns long and twenty times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.


A Ligand-reversible Dimerization System For Controlling Protein-protein Interactions

CT Rollins, VM Rivera, DN Woolfson, T Keenan, M Hatada, SE Adams, LJ Andrade, D Yaeger, MR van Schravendijk, DA Holt, M Gilman, and T Clackson

Proc. Natl. Acad. Sci. U.S.A. 97, 7096-7101 (2000) DOI: 10.1073/pnas.100101997

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (FM) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving FM fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated FM domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.


Core-directed Protein Design. I. An Experimental Method For Selecting Stable Proteins from Combinatorial Libraries

MD Finucane, M Tuna, JH Lees, and DN Woolfson

Biochemistry 38, 11613-11623 (1999) DOI: 10.1021/bi990765n

The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive folding and stabilisation of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterisation of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows: hexahistidine-tagged proteins were displayed on the surface of phage; these protein-phage were immobilised onto Ni1-coated surfaces; the bound fusion-phage were treated with protease to remove unstable or poorly folded proteins; stable phage fusions were eluted and infected into E. coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatised supports were tested: Ni-NTA chips for surface plasmon resonance (SPR), and Ni-NTA agarose beads. SPR carried the advantage that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was done using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterisation of which is described in the accompanying paper (Finucane & Woolfson, accompanying paper). As our method selects based only on structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.


Core-directed Protein Design. II. Rescue of a Multiply Mutated and Destabilized Variant of Ubiquitin

MD Finucane and DN Woolfson

Biochemistry 38, 11604-11612 (1999) DOI: 10.1021/bi990766f

We have applied the method described in the accompanying paper (Finucane et al., accompanying paper), namely stability-based selection using phage display, to explore the sequence requirements for packing in the hydrophobic core of ubiquitin. In contrast to the parent protein, which was a structurally compromised mutant, the selected variants could be overexpressed and purified in yields for structural studies. In particular, CD and NMR measurements showed that the selectants folded correctly to stable native-like structures. These points demonstrate the utility of our core-directed method for stabilising and redesigning proteins. In addition and in contrast to foregoing studies on other proteins, which suggest that hydrophobic cores permit substitutions provided that hydrophobicity and core volumes are generally conserved, we find that the core of ubiquitin is surprisingly intolerant of amino-acid substitutions; variants that survived our selection showed a clear consensus for the wild-type sequence. It is probable that our results differed from those from other groups for two reasons: first, ubiquitin may be unusual in that it has strict sequence requirements for its structure and stability. We discuss this result in light of sequence conservation in the eukaryotic ubiquitins and proteins of the ubiquitin structural superfamily. Second, our mutants were selected solely on the basis of stability, in contrast to the other studies that rely on function-based selection. The latter may lead to proteins that are more plastic and tolerant of substitutions.


Regulation of Hsp90 ATPase Activity by Tetratricopeptide Repeat (TPR)-domain Co-chaperones

C Prodromou, G Siligardi, R O'Brien, DN Woolfson, L Regan, B Panaretou, JE

EMBO J. 18, 754-762 (1999) DOI: 10.1093/emboj/18.3.754

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 crith yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90, Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co- chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90, Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90, These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP- dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.


Determinants of Strand Register in Antiparallel Beta-sheets of Proteins

EG Hutchinson, RB Sessions, JM Thornton, and DN Woolfson

Protein Sci 7, 2287-2300 (1998) DOI: 10.1002/pro.5560071106

Antiparallel beta-sheets present two distinct environments to inter- strand residue pairs: beta(A,HB) sites have two backbone hydrogen bands; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only similar to 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic- Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi(1) conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi(1) geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche(-) conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de nova design. Our findings are discussed in light of previous analyses and experimental studies.


Coiled-coil Assembly by Peptides With Non-heptad Sequence Motifs

MR Hicks, DV Holberton, C Kowalczyk, and DN Woolfson

Fold. Des. 2, 149-158 (1997) DOI: 10.1016/S1359-0278(97)00021-7

Background: The seven-residue heptad repeat is the accepted hallmark of coiled coils. In extended filamentous proteins, however, contiguous patterns of heptads are often disrupted by 'skips' and 'stammers'. The structural consequences and roles of these digressions are not understood. Results: In a cytoskeleton protein from Giardia lamblia, heptads flank eleven-residue units (hendecads) to give a 7-11-7 motif that dominates the sequence. Synthetic peptides made to the consensus sequence of this motif fold in solution to fully helical, parallel dimers. Both the sequence pattern and these experimental data are consistent with the coiled-coil model, We note that breaks in other extended coiled coils can also be reconciled by hendecad insertions, Conclusions: The heptad paradigm for the coiled coil must be expanded to include hendecads. As different combinations of heptads and hendecads will give different overall sequence motifs, we propose that these provide a mechanism to promote cognate protein pairings during the folding of extended coiled coils in the cell.


Buried Polar Residues and Structural Specificity in the GCN4 Leucine Zipper

L Gonzalez, DN Woolfson, and T Alber

Nat. Struct. Biol. 3, 1011-1018 (1996) DOI: 10.1038/nsb1296-1011

A conserved asparagine (Asn 16) buried in the interface of the GCN4 leucine zipper selectively favours the parallel, dimeric, coiled-coil structure. To test if other polar residues confer oligomerization specificity, the structural effects of Gln and Lys substitutions for Asn 16 were characterized. Like the wild-type peptide, the Asn16Lys mutant formed exclusively dimers. In contrast, Gln 16, despite its chemical similarity to Asn, allowed the peptide to form both dimers and trimers. The Gln 16 side chain was accommodated by qualitatively different interactions in the dimer and trimer crystal structures. These findings demonstrate that the structural selectivity of polar residues results not only from the burial of polar atoms, but also depends on the complementarity of the side-chain stereochemistry with the surrounding structural environment.


A Designed Heterotrimeric Coiled-coil

S Nautiyal, DN Woolfson, DS King, and T Alber

Biochemistry 34, 11645-11651 (1995) DOI: 10.1021/bi00037a001

Principles that guide folding of coiled coils were tested by designing three peptides that preferentially associate with each other to form a heterotrimeric coiled coil. The core positions of the designed helices contained residues that promote formation of trimeric coiled coils. Ionic interactions were employed to mediate heterospecificity, and negative design was used to favor formation of the heterotrimer over alternative arrangements. A program was written to select sequences that maximized the number of attractive interhelical interactions in a parallel heterotrimer and the number of repulsive electrostatic interactions in alternative species. Solution studies indicate that an equimolar mixture of the three peptides forms a helical trimer with high specificity and stability. These results validate the principles used to guide the design and suggest that the heterotrimer may serve as a useful, autonomous trimerization domain.


Predicting Oligomerization States of Coiled Coils

DN Woolfson and T Alber

Protein Sci. 4, 1596-1607 (1995) DOI: 10.1002/pro.5560040818

An algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two- and three-stranded, coiled-coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations-profile values of solvent-exposed residues and of amino acids that were underrepresented in the database were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.


Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin.

JPL Cox, PA Evans, LC Packman, DH Williams, and DN Woolfson

J. Mol. Biol. 234, 483-492 (1993) DOI: 10.1006/jmbi.1993.1600

The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal ?-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an ?-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the 1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the ?-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the ?-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.


Topological and Stereochemical Restrictions in Beta-sandwich Protein Structures

DN Woolfson, PA Evans, EG Hutchinson, and JM Thornton

Protein Eng. 6, 461-470 (1993) DOI: DOI: 10.1093/protein/6.5.461

Chain topology in beta-structured protein domains and handedness associated with it are discussed. Previously, other workers have shown that by considering just two restrictions-structures that are left-handed and/or have loops that cross can be disregarded-the number of topologies associated with such structures is expected to be severely limited. By way of example, we determine the number of topologies compatible with a six-stranded antiparallel beta-sandwich. Without restriction on the type of strand - strand connection allowed but with elimination of symmetry related structures 360 topologies are possible. If connections between parallel strands are disqualified the number is reduced, 10-fold, to 36. The figure is cut to 24 when structures with loop crossings are eliminated. Handedness in these structures is examined in detail and from this a rationale for the observed predominance of right-handed forms of beta- structures is presented. The 24 structures can be considered as a set of right- and left-handed pairs of 12 topologies. All but two of these pairs can be assigned hands on the basis of existing rules. Six of the structures are found to occur in the Brookhaven Protein Databank and all are right-handed. This study provides a basis for protein design projects which might, for example, attempt the synthesis of unobserved protein topologies. Of the 24 structures in the final set eight are examples of the classic Greek key fold. Thus, the predominance of this motif among all-beta proteins can be attributed in part to these topological constraints. The possible physicochemical origins of the structural selection rules and additional factors which might contribute to the particular favourability of certain structures are also explored.


Protein Folding in the Absence of the Solvent Ordering Contribution to the Hydrophobic Interaction

DN Woolfson, A Cooper, MM Harding, DH Williams, and PA Evans

J. Mol. Biol. 229, 502-511 (1993) DOI: 10.1006/jmbi.1993.1049

Despite considerable effort there is no consensus as to what interaction, or set of interactions, provides the dominant force that drives protein folding and specifies folded protein structures. A key thermodynamic observation is that a large drop in heat capacity (delta Cp) usually accompanies folding in water. Various factors may contribute to this effect, especially changes in the structure of the solvent upon exposure of both non-polar and polar groups in the unfolded state. The unfavourable Gibbs free energy of solvating non-polar groups, in particular, is thought to provide a central driving force for folding (the hydrophobic effect) but the role of solvent ordering in this remains a matter of controversy. We report here a series of experiments that show that a protein can fold into its native conformation under conditions where solvent ordering effects are demonstrably negligible. In methanol/water mixtures ubiquitin unfolds reversibly with a delta Cp value that falls close to zero above about 30% (v/v) methanol. We are able to reason, on the basis of these data, that the net contribution to the heat capacity change arising primarily from the protein structure itself is not significant and that contributions from changes in solvent ordering are rendered negligible by the change in composition. Nuclear magnetic resonance measurements, however, indicate that non-polar side-chains do still become exposed to solvent in the denatured state under these conditions. The combination of these results and model compound studies suggests that the elimination of ordering effects is an intrinsic property of the mixed solvent. We can, therefore, conclude that the solvent ordering component of the hydrophobic effect is not an obligatory factor in determining the three-dimensional structure into which the protein will fold.


Depicting Topology and Handedness in Jellyroll Structures

HJ Stirk, DN Woolfson, EG Hutchinson, and JM Thornton

FEBS Lett. 308, (1) 1-3 (1992) DOI: 10.1016/0014-5793(92)81036-L

The jellyroll structure is a special case of the Greek key topology and, to date, has only been observed in complete form in one of its four possible arrangements. Like other elements of super-secondary structure involving the beta-strand (e.g. the beta-alpha-beta unit) the known structure forms a right-handed superhelix. The possibility of losing such tertiary information and other problems associated with representing these structures by two-dimensional topology diagrams are discussed. A series of rules are presented which allow this three-dimensional information to be represented in two- dimensional topology diagrams from which the handedness of a jellyroll structure can be determined.


Conserved Positioning of Proline Residues in Membrane-spanning Helices of Ion-channel Proteins

DN Woolfson, RJ Mortishiresmith, and DH Williams

Biochem. Biophys. Res. Commun. 175, 733-737 (1991) DOI: 10.1016/0006-291X(91)91627-O

No Abstract Available


Characterization of a Partially Denatured State of a Protein by 2- Dimensional NMR Reduction of the Hydrophobic Interactions in Ubiquitin

MM Harding, DH Williams, and DN Woolfson

Biochemistry 30, 3120-3128 (1991) DOI: 10.1021/bi00226a020

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta- sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha- helix in the N-state. There is evidence for some fixed side-chain- side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.


A 3-disulfide Derivative of Hen Lysozyme. Structure, Dynamics and Stability.

SE Radford, DN Woolfson, SR Martin, G Lowe, and CM Dobson

Biochem. J. 273, 211-217 (1991) DOI: 10.1042/bj2730211

A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two- dimensional H-1-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three- disulphide lysozyme (CM6,127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6,127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6,127- lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6,127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability.


Hydrophobic Clustering in Nonnative States of a ProteinInterpretation of Chemical-shifts in NMR-spectra of Denatured States of Lysozyme

PA Evans, KD Topping, DN Woolfson, and CM Dobson

Proteins 9, 248-266 (1991) DOI: 10.1002/prot.340090404

Chemical shifts of resonances of specific protons in the H-1 NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.


The Influence of Proline Residues on Alpha-helical Structure

DN Woolfson and DH Williams.

FEBS Lett. 277, 185-188 (1990) DOI:10.1016/0014-5793(90)80839-B

Proline lacks an amide proton when found within proteins. This precludes hydrogen bonding between it and hydrogen bond acceptors, and thus often restricts the residue to the first four positions of an alpha-helix. Helices with proline after position four have a pronounced kink [(1988) J. Mol. Biol. 203, 601-619]. In these cases, we find that the proline residue almost always occurs on the solvent exposed face of each helix. This positioning facilitates the compensatory hydrogen bonding between solvent and residues P-3 and P- 4 (relative to proline, P), through the formation of the kink. Further, it aids in the packing of long helical structures around globular protein structures.