Peptide co-assembly, in which complementary molecules form nanofibers when combined yet remain unassembled when alone, is emerging as a simple and versatile approach to create functional supramolecular materials for various biomedical and biotechnological applications. Charge complementarity provides a simple route to design selectively co-assembling peptide pairs, yet detailed understanding of the influence of net charge on the assembly process remains limited. Here we report a method to understand charge-complementary peptide co-assembly that combines molecular modeling, biophysical characterization, and solid-state NMR structure analysis. Specifically, we designed a set of peptides, CATCH(X+) or CATCH(X-), with the general sequence (C/Q)-Q-(C/Q)-F-(C/Q)-F-(C/Q)-F-(C/Q)-Q-(C/Q) where X denotes net charge at neutral pH and C denotes either E or K. Secondary structure modeling using PRIME20 predicted that all complementary CATCH pairs would co-assemble into β-sheet rich structures with an alternating +/-/+/- arrangement. In contrast, CATCH(4-), CATCH(6-), and CATCH(6+) would be resistant to self-assembly, while CATCH(4+) would have weak self-assembly propensity. Consistent with these models, elongated fibrils with widths of ~5 nm were observed in samples of all complementary CATCH pairs visualized using transmission electron microscopy, whereas no fibrils were observed in samples of any CATCH peptide alone. Spectroscopic analysis indicated that complementary CATCH peptide pairs co-assembled into structures rich in β-sheets, whereas individual CATCH peptides adopted a random coil configuration. Thioflavin T (ThT) fluorimetry largely confirmed these observations, although CATCH(4+) demosntrated propensity for weak self-assembly consistent with PRIME20 models. Finally, solid-state NMR of CATCH(6+/4-) assemblies demonstrated that these peptides formed β-sheet nanofibers with a predominantly alternating +/-/+/- arrangement, consistent with PRIME20 predictions. Interestingly, ThT fluorimetry and discontinuous molecular dynamics suggested that CATCH(6+/6-) would co-assemble faster than the (4+/4-) pair, suggesting a role for long-range electrostatic attraction. Together, these data demonstrate that molecular modeling, biophysical characterization, and solid-state NMR structure analysis can provide key insights into the process of peptide co-assembly that will be useful for establishing sequence design rules for this emerging class of supramolecular biomaterials.

Understanding charge-complementary peptide co-assembly using a computational-experimental framework

2019 Spring National Meeting

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