Poster O5 - Substituent Effects on pi-Stacking and Self-assembly of Phenylalanine-rich Amphipathic Peptides

Introduction: De novo peptides composed of alternating phenylalanine (Phe, F) and charged amino acids self-assemble into cross-β rich amyloid-like nanofibrils. The self-assembly of these peptides is guided by noncovalent interactions composed of electrostatic bridges, hydrogen bonds, and hydrophobic and van der Waals interactions. Subtle changes in these balancing forces can drastically alter the structure and morphology of the nanofibril assemblies and bulk properties of the resulting hydrogels. In this work, we utilized the pentapeptide KFE5 (Ac-FKFEF-Am) to determine how substitution of electron donating (-OH, -CH3) or withdrawing (-NO2, -CN, -Br) groups at the para position of the Phe ring impacts self-assembly, packing, and biophysical properties of the resulting nanostructures (Fig. 1A). We also explored the effect of replacing the Phe in KFE5 with Homophenylalanine (FH) or Phenylglycine (FG), whose side chains are one carbon bond longer or shorter than F respectively (Fig. 1A). Our data indicates significant effects of the substitutions on the morphology and bulk properties of the resulting nanofibrils and hydrogels.

Materials and

Methods: All peptides were synthesized using standard Fmoc-chemistry and purified (>90%). The β-sheet content of the fibrils was determined by Thioflavin T (ThT) assay. Transmission electron microscopy (TEM) was used to visualize fibril morphology. Secondary structures were determined using circular dichroism (CD) spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). To assess cross seeding, α-syn biosensor cells were seeded with α-syn preformed fibrils or with modified KFE5 fibrils at various concentrations and FRET signal was quantified. Cytotoxicity was assessed using MTT assays and bulk gel formation was characterized using parallel plate rheology. The effects of the substitutions on innate immune cells was tested by treating dendritic cell (DC) cultures with the peptide fibrils and analyzing the release of chemokines and cytokines released in multiplex assays.

Results, Conclusions, and Discussions: Results and Discussions: Electron microscopy data indicated that KFE5 assembled into broad tapes compared to the well-studied KFE8 (FKFEFKFE) which assembles into thin 8-10 nm wide fibrils. While the OH- substitution led to complete abrogation of assembly, Br- substitution resulted in wide flat tapes and CH3 substitution resulted in the formation of twisted tapes (Fig. 1C). Substitution with CN- and NO2- resulted in ultra-thin fibrils with high aspect ratio. The FH variant formed twisted tapes of uniform width, while the FG substituted peptide formed thinner and shorter fibrils (Fig 1C). Secondary structure analysis of all assemblies (with the exception of OH-) displayed a characteristic minimum at 218 nm, indicative of β-rich structures (data not shown), which was further confirmed using Thioflavin-T (ThT) assay (Fig. 1B). All peptides were found to be non-toxic in mammalian cell cultures (data not shown). We further demonstrate that preformed fibrils of all peptides do not cross-seed α-synuclein in an α-synuclein FRET biosensor cell line (Fig. 1D). Assessing the gelation properties of the peptides revealed that only those peptides with an electron withdrawing groups on Phe were able to form self-supporting gels (Fig. 1E), suggesting that the distribution of electron density on the phenyl ring has a significant impact on the assembly of amphipathic peptides.

Conclusions: Our findings confirm that distribution of electron density on the phenyl ring has a significant impact on the assembly of amphipathic peptides and can be used as a design tool to tune the molecular packing and bulk properties of the resulting structures. This provides useful insights into their applications as scaffolds for biomedical applications.

Acknowledgements (Optional): This work was funded in part by NIH/NIA R21AG06873302 to M.E.J and J.S.R.