Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although alpha-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of alpha-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative beta-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

Automated summary

Using molecular dynamics simulations and experimental studies, it was found that double-fragment peptide fibrils have lower relative beta-sheet content compared to single-fragment peptide fibrils. The morphological differences, such as short protofibrils, elongated, twisted, and rod-like structures, in non-capped and capped double-fragment peptide systems were confirmed through TEM analysis. The differences in Thioflavin T fluorescence lifetime profiles upon fibril binding suggest potential morphological differences in the fibril samples.