Abstract
Determining the structure of the (oligomeric) intermediates that form during the self‐assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo‐induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid‐forming peptide, Aβ16‐22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group—Fmoc(TFMD)Phe—was incorporated into the sequence (Aβ*16–22). Electrospray ionization ion‐mobility spectrometry mass‐spectrometry (ESI‐IMS‐MS) analysis of the PIC products confirmed that Aβ*16–22 forms assemblies with the monomers arranged as anti‐parallel, in‐register β‐strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aβ16‐22 assembles through a single‐step nucleation into a β‐sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aβ16‐22 self‐assembly processes, paving the way to explore the self‐assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.
Highlights
Hall and co-workers[23] applied discontinuous molecular dynamics (DMD) and a coarse-grained protein model (PRIME20) to demonstrate that at high simulation temperatures, a system of 48 monomeric Aβ16-22 peptides aggregates via classical nucleation and growth into a highly-ordered structure with monomers organized as anti-parallel, in-register β-strands in agreement with the solid-state NMR measurements reported by Tycko and coworkers.[5]
Aβ16-22, a TAMRA-labeled Aβ16-22 peptide variant and an Aβ*16–22 peptide variant were synthesized and used in fluorescence quenching and photo-induced crosslinking (PIC) assays to demonstrate that Aβ16-22 aggregates in two distinct phases
PIC with ESI-IMS-MS/MS at different time points confirmed that Aβ*16–22 forms assemblies in which monomers are organized as anti-parallel, in-register β-strands at all time points
Summary
Hall and co-workers[23] applied discontinuous molecular dynamics (DMD) and a coarse-grained protein model (PRIME20) to demonstrate that at high simulation temperatures, a system of 48 monomeric Aβ16-22 peptides aggregates via classical nucleation and growth into a highly-ordered structure with monomers organized as anti-parallel, in-register β-strands in agreement with the solid-state NMR measurements reported by Tycko and coworkers.[5] At lower simulation temperatures, Aβ16-22 first forms β-sheetrich oligomers which merge and rearrange into a large fibril. The results highlight the power of combining PIC with ESI-IMS-MS/MS, fluorescence quenching and DMD simulations to study kinetic intermediates in peptide self-assembly, and paves the way for exploring the self-assembly of larger, more complex peptides, including those directly relevant to amyloid disease
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