Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aß(1-42) in Amyloid Fibrils.

ABSTRACT

Intense efforts have been made to understand the molecular structures of misfolded amyloid ß (Aß) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aß peptide Aß(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22499-505, 2015). Despite great biological and clinical interest in Aß(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aß(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aß(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aß(1-42) fibrils as seeds. Next, we detail analysis of Aß(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aß(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.