Design of parallel 𝛽-sheet nanofibrils using Monte Carlo search, coarse-grained simulations, and experimental testing.

Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel β-sheets", that is, β-sheets with N-terminus-to-C-terminus 𝛽-strand vectors oriented in parallel. To accomplish the desired β-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-β spine defined by Sawaya et al. This molecular configuration includes two layers of parallel β-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent β-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel 𝛽-sheet arrangement, two formed fibril-like structure with <50% in parallel 𝛽-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel β-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.