(B-58) Divergent Suprastructures of Self-Assembling Block Heterochiral Peptides

Introduction:: De novo-designed oligopeptides that self-assemble have diverse applications in engineering and medicine, aided by their ease of synthesis and customization¹. The amphipathic peptide KFE8 (Ac-FKFEFKFE-NH₂) with alternating hydrophobic and hydrophilic residues forms highly soluble fibrils in water. We have previously reported that the inclusion of both L- and D-chiral FKFE repeat blocks in the same sequence – termed block heterochirality – generates fibrils with divergent morphologies. This work describes the expansion of this concept to a triblock system KFE12, which gives rise to more diverse structures. Understanding the fundamental tendencies and general behavior of these peptides is important not just to characterize their self-assembly, but also to maximize the effectiveness of their biological applications.

Materials and Methods:: Transmission electron microscopy (TEM) and scanning TEM (STEM) were used to visualize fibril morphology. The widths and helical pitches were quantified using FIJI software. Secondary structures were determined using circular dichroism (CD) spectroscopy, small- and wide-angle x-ray scattering (SAXS and WAXS), and Fourier transform infrared spectroscopy (FT-IR). Proposed packing modes were informed by energy-minimized molecular dynamics (MD) models that match observed dimensions and scattering data. Parallel-plate rheometry was performed on peptide gels to examine bulk material properties. All peptides were acquired from P3 BioSystems and used without further purification.

Results, Conclusions, and Discussions:: It has been previously shown that the LL and DD analogs of KFE8, with the two-letter codes referring to the chirality of their FKFE repeat blocks, assemble into cross-β nanofibers having widths of 8 ± 0.3 nm while the heterochiral analogs of KFE8, LD and DL, are an order of magnitude wider, with measured values of ~108 ± 55 nm². Further morphological variation was seen upon the expansion of block heterochirality from KFE8 to KFE12. All KFE12 analogs form bilayer β-sheets and enantiomeric pairs (e.g., LLL and DDD) form identical assemblies aside from handedness. LLL (Fig 1A) and DDD form short fibrils that are one peptide across (~4.7, ~4.9 nm). LDL (Fig 1B) and DLD assemblies are twisted ribbons with a large pitch (~300 nm) and a width of three β-sheets (~15.6, ~17.2 nm). LLD (Fig 1C) and DDL peptides form two-β-sheet wide ribbons without apparent helicity (~7.5, ~6.9 nm). LDD (Fig 1C) and DLL assemble into amorphous β-sheets complexes from smaller, seemingly 2-D fragments to micron-scale 3-D aggregates. These analogs are further characterized through CD, FT-IR, SAXS, WAXS, and rheology. CD traces for enantiomeric pairs were mirrored, reaffirming the visual similarities in morphology that were observed via EM, while FT-IR verified that all analogs formed antiparallel β-sheets. X-ray scattering analysis showed that all KFE12 analogs had similar underlying packing modes and that LDD and DLL have a larger diversity in structural features. Rheological data show that all peptide gels display shear thinning behavior, except for LDD and DLL whose tendency to form large aggregates prevented formation of self-supporting gels. The extension from di- to triblocks revealed that block heterochirality can generate additional unique morphologies from KFE12. Further investigation of these diverse structures will shed light on fundamental aspects of self-assembly, while in vivo and in vitro studies may inform and enable modulation of the KFE systems’ previously established immunological applicability.

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References (Optional): : 1) Bowerman CJ. et al. Pept Sci. 2012. 98: 169-184.
2) O’Neill CL. et al. 2020. J. Am. Chem. Soc. 2020, 142, 47, 19809-19813.