title: The role of prefibrillar structures in the assembly of a peptide amyloid.
abstract: The self-assembly of proteins into stable, fibrillar aggregates is a general property of polypeptides most notably associated with degenerative diseases termed amyloidoses. These nano- to micrometer scale structures are formed predominantly of beta-sheets that self-assemble by a nucleation-dependent mechanism. The rate-limiting step of assembly involves stabilization of high-energy intermediates in a kinetic step termed nucleation. Determination of the structural characteristics of these high-energy intermediates has been elusive, as its members are the least populated states on the assembly pathway. Using a peptide derived from diabetes-related amyloid, we use electron paramagnetic resonance (EPR) spectroscopy and disulfide crosslinking to show that fibers are composed of parallel, in-register beta-sheets. Kinetic studies are then used to infer the structural elements of the pre-nucleation intermediates. Notably, stabilization of this ensemble is shown to depend on the number but not the position of amide side chains within the primary sequence. Additionally, fiber formation is accelerated by constructs that mimic the intra-sheet structure of the fiber. Our data suggest that pre-nucleation intermediates sample intra- beta-sheet structure and place bounds on the possible nucleation mechanisms for fiber assembly. Understanding the nucleation of fibrillogenesis is critical so that this process can be prevented in disease and productively controlled by design.
authors.affiliation: Department of Chemistry, Yale University, 350 Edwards Street, New Haven, CT 06520-8107, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Ruschak
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Miranker
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008954:Models, Biological; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D000682:Amyloid; D004220:Disulfides