Supplementary MaterialsSupplementary Figures. raise possibilities for using the hSAFs as substrates

Supplementary MaterialsSupplementary Figures. 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. As our understanding of sequence-to-structure associations in proteins improves, so does our ability to rationally design new proteins and protein-based materials. Unlike discrete peptide and protein objects, the design of biomaterials KW-6002 manufacturer requires additional rules for self-assembly to allow the nano-to-micron scale regimes to be bridged 2, 4. In these respects, synthetically accessible peptideswhich can be programmed to fold into prescribed structures, and to self-assemble into larger architecturesoffer routes to rationally designed peptide and protein-based biomaterials. Indeed, a variety of peptide-based self-assembling fibres, tapes and hydrogels have been produced 6-11. Much of this effort has been directed to the assembly of -structured systems, though -helix-based fibrous and -helix-containing gelling materials have been explored to some extent8, 12-17. Previously, we have described a number of fibrous biomaterials based on the -helical coiled coil8, 18, 19. These so-called SAFs (self-assembling fibers) comprise two 28-residue peptides designed to co-assemble, resulting in an offset -helical dimer with complementary sticky ends. The ends promote longitudinal assembly into -helical coiled-coil fibrils, which bundle to form matured fibers. Elsewhere, we have demonstrated that specific interactionsfostered by features on the surface of the coiled coillead to crystallization of the peptides within the fibers, Physique 1a.19 Consequently, fibers are highly ordered, thickened, and settle out of Rabbit Polyclonal to HOXD8 solution. For the work presented here, we reasoned that replacing these few specific interactions with many more-general interactions would lead to networks of non-covalently cross-linked fibrils and, hence, physical hydrogels3, Physique 1b. Open in KW-6002 manufacturer a separate KW-6002 manufacturer window Physique 1 hSAF design principles(a) In previous SAF designs, specific charged interactions between certain b and c positions lead to peptide alignment and fiber thickening. (b) For the hSAFs, we replaced these specific interactions with weaker, more-general interactions at all and sites, to result in smaller, more flexible, bundles of thinner fibres. The design rules for the hydrogelating SAF (hSAF) peptides initially followed those for the SAFs8. That is, they were two-component systems based on the coiled-coil heptad sequence repeat, and positions are responsible for directing the dimer interface, Figure 1, these were maintained from the original SAF design. Where the hSAFs differ from the original designs is at the and positions, which are exposed KW-6002 manufacturer around the surfaces of the coiled-coil assemblies. For the hSAFs these positions were made combinations of alanine and glutamine residues: alanine was chosen to promote poor hydrophobic interactions between fibrils; and glutamine for its propensity to hydrogen bond. Initially, three hSAF designs were investigated: variants hSAFAAA, hSAFQQQ and hSAFAAQ, Table 1, where subscripts denote amino acids at and and with the larger glutamine, and (2) the likely additional solvation of these hydrophilic residues. CD spectra and XRD patterns, consistent with these assertions were obtained for the hSAFAAQ control fibres, (Physique S4, Supporting Information). To probe the power of the hSAFAAA gels as a substrate for cell growth, we tested for peptide cytotoxicity and cell differentiation using rat adrenal pheochromocytoma (PC12) cells. First, however, we had to further stabilize the fibril-fibril interactions and the resulting gels. This was because, though hSAFAAA gels could be washed and soaked in both phosphate-buffered saline (PBS) and standard cell-culture media, they did not persist for sufficient time to allow sustained cell-culture experiments. To stabilize the gels, in each of the hSAFAAA peptides we replaced one of the surface-exposed alanine residues at an position with the more-hydrophobic tryptophan, Table 1. This also allowed easy quantification of peptide concentration. In all respectsspectroscopic, microscopic and gel formationthe hSAFAAA-W combination behaved similarly to the parent peptides (Physique S5 Supporting Information). Moreover the new peptides gelled at room temperature and the gels were stable in PBS and cell-culture media at 37 C for more than two weeks, which permitted cell-biology studies as follows. In Alamar Blue cell-proliferation assays24, PC12 cells seeded on collagen and then treated with increasing concentrations (0.5 – 2.5 mM, equivalent to 1.5 – 7.5 mg/ml, total pepide) of hSAFAAA-W peptides and gels proliferated, and were statistically no different to controls.