Patrick King's Research Page

Previous work

Biomineralisation of SAFs

This work was undertaken in 2005/2006 as a research project in the final year of my undergraduate degree at Bristol, working in a collaboration between the Woolfson and Mann labs (working closely with David Papapostolou and Stewart Holmström). This project involved the biomineralisation of matured SAFs. Two materials were used, an organically functionalised 2:1 magnesium:phyllosilicate clay and silica. One reason for biomineralisation was to try to increase the SAFs' stability under physiological conditions. It was found that silica was the best at increasing fibre stability with respect to temperature, pH and salt concentration. Hollow silica nanotubes were also formed by using the SAFs as a sacrificial template for silica deposition, using silicic acid. Further work is ongoing.

New (oct 2009)

The SAF system

The rational design of peptide-based fibrous systems has become an important and growing field within materials research due to their potential use as tissue engineering scaffolds. In 2000 our research group reported the rational design of the self-assembling fibre (SAF) system(1), which comprises two leucine-zipper peptides designed to form sticky ended dimers that propagate into long non-covalently bound α-helical coiled-coil fibrils (figure 1). A combination of biophysical techniques has revealed mature SAFs to be an average of ∼40 microns in length and ∼70 nm in width, with a high level of internal order(2). Rational peptide redesign has led to SAF systems that demonstrate improved stability(3), altered morphology(2,4), and that have been shown to produce hydrogels for use in 3D tissue culture(5).

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 1: SAF peptides are designed de novo to form sticky-ended coiled coils, which propagate into fibres. SAF peptide sequences are shown, alongside a helical wheel diagram to show the positioning of these residues in the coiled-coil. NB: coiled-coils also laterally assemble to form structures ∼40 µm in length and ∼70 nm in width.

SAF assembly pathway

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 2: A diagrammatic representation of the assembly pathway through which SAFs assemble. Initially SAF peptides form a dimeric species via a coiled-coil interaction, and further associate to form a dimer/tetramer equilibrium. These species further assemble to form a nucleus that seeds the growth of fibres. Growth then proceeds in 3 dimensions, until later stages, when fibre growth becomes predominantly epitaxial. Fibres continue to grow until peptide concentration falls below the level that tetrameric peptide assemblies form.

Investigation into the pathway through which the SAF system assembles has furthered our understanding of how the system can be manipulated to improve future designs and allow greater control over the functionalisation of the system. To this end, a combination of biophysical techniques have been used to investigate the SAF assembly pathway. Circular dichroism spectroscopy, NMR spectroscopy, transmission electron microscopy, and fluorescence microscopy have revealed that SAFs form via a nucleation and growth mechanism (figure 3).

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 3: Kinetics of SAF assembly followed using circular dichroism and nuclear magnetic resonance spectroscopy. The presence of a lag phase and shape of the kinetic profile implies a nucleation and growth mechanism occurs during SAF assembly.

Assembly initially occurs in three dimensions, until an hour into assembly, after which fibre growth is predominantly epitaxial. The introduction of ‘terminator’ mutants that cannot assemble into fibres has allowed isolation of the initial folding of the system, which has been characterised using circular dichroism spectroscopy and analytical ultracentrifugation (figure 4).

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 4: Comparison of the CD spectrum of mixed SAF peptides with the CD spectrum of terminator peptide mixtures. Terminator peptide mixtures which mimic SAF peptides binding to one another via their non-asparagine-containing regions mimic the initial CD spectrum of the SAF system before assembly occurs. Because terminator peptides prevent the assembly of fibres, analytical ultracentrifugation could be performed to characterise the assemblies formed. At below the critical concentration for fibre formation, a monomer/dimer equilibrium was observed, whereas at concentrations at which SAFs form and above, a dimer/tetramer equilibrium was observed.

Manipulation of the SAF system

The system has been rationally manipulated to vary fibre length through seeding of SAF peptide mixtures with SAF fragments (figure 5)

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 5: Pre-formed SAFs were broken apart and used to seed the assembly of a freshly mixed SAF sample. Addition of a smaller number of seeds led to the formation of fewer, longer fibres. Addition of more seeds led to the formation of a large number of small fibres.

Fibres have been observed to assemble in a non-polar manner using fluorescence microscopy. However, polar assembly was induced by the addition of terminator peptide species during SAF assembly which block assembly at a specified end of the fibre (figure 6).

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 6: Top: A diagrammatic representation of the proposed action of terminator species at fibre ends to prevent further assembly in that direction. Terminator species can bind only to one fibre end, but are not sticky-ended and so fibres cannot continue to assemble at the blocked end. Bottom: A comparison of fibres formed with and without the inclusion of terminator species. SAFs formed without terminator have a javelin morphology (having grown from both fibre ends), whereas SAF grown in the presence of terminator species have a snooker-cue morphology, and are on average half the length of SAFs grown without the presence of terminator species.

SAF biomineralisation(1)

SAFs were found to be ideal templates for the nucleation and growth of silica from an aqueous solution of Si(OH)4. SAFs were controllably coated in silica to form organic/inorganic hybrid materials. Kinked and segmented SAFs(7) were also functionalised in this manner. Organic material was removed using harsh conditions or, preferentially, using an enzyme such as chymotrypsin to leave hollow silica tubes. Both materials were further functionalised by deposition of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride).

SAF-p1 and SAF-p2 assemble to form a sticky ended dimer

Figure 7: TEM micrographs illustrating the synthesis of silica nanotubes on peptide templates with various morphologies. Key: column 1, self-assembled peptide scaffolds formed by mixing specified peptides and overnight incubation at 20 °C. Column 2, silicified peptide scaffolds. Column 3, silica replicas left after proteolysis of the protein cores with chymotrypsin. Row 1, standard linear SAFs. Row 2, segmented morphologies. Row 3, kinked morphologies.

Publication record

[1] 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).

[2] 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).

[3] The assembly pathway of a design α-helical protein fiber. EHC Bromley KJ Channon, PJS King, ZN Mahmoud, EF Banwell, MF Butler, MP Crump, TR Dafforn, MR Hicks, JD Hirst, A Rodger and DN Woolfson, Submitted to PNAS (2009).


[4] M.J. Pandya et al., Biochemistry, 39, 8728-8734 (2000).

[5] D. Papapostolou et al., Proc. Natl. Acad. Sci. U. S. A., 104, 10853-10858 (2007).

[6] A.M. Smith et al., Adv. Func. Mat., 16, 1022-1030 (2006).

[7] M.G. Ryadnov and D.N. Woolfson, J. Am. Chem. Soc., 127, 12407-12415 (2005).

[8] E.F. Banwell et al., Nat. Mater., 8, 596-600 (2009).