A lowly populated, transient β-sheet structure in monomeric Aβ identified by multinuclear NMR of chemical denaturation.

Chemical denaturation is a well-established approach for probing the equilibrium between folded and unfolded states of proteins. We demonstrate applicability of this method to the detection of a small population of a transiently folded structural element in a system that is often considered to be intrinsically fully disordered. The H, N, C, and C' chemical shifts of Aβ and Aβ peptides and their M35-oxidized variants were monitored as a function of urea concentration and compared to analogous urea titrations of synthetic pentapeptides of homologous sequence. Fitting of the chemical shift titrations yields a 10 ± 1% population for a structured element at the C-terminus of Aβ that folds with a cooperativity of m = 0.06 kcal/mol·M. The fit also yields the chemical shifts of the folded state and, using a database search, for Aβ these shifts identified an antiparallel intramolecular β-sheet for residues I32-A42, linked by a type I' β-turn at G37 and G38. The structure is destabilized by oxidation of M35. Paramagnetic relaxation rates and two previously reported weak, medium-range NOE interactions are consistent with this transient β-sheet. Introduction of the requisite A42C mutation and tagging with MTSL resulted in a small stabilization of this β-sheet. Chemical shift analysis suggests a C-terminal β-sheet may be present in Aβ too, but the turn type at G37 is not type I'. The approach to derive Transient Structure from chemical Denaturation by NMR (TSD-NMR), demonstrated here for Aβ peptides, provides a sensitive tool for identifying the presence of lowly populated, transiently ordered elements in proteins that are considered to be intrinsically disordered, and permits extraction of structural data for such elements.