An NMR-Based Quenched Hydrogen Exchange Investigation of Model Amyloid Fibrils Formed by Cold Shock Protein A

Abstract

Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (~1 x 10-3 min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10-5 min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are ~10 3 , and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10 5 on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14

“…9B). Similar findings, where almost the entire peptide/protein was observed to be structured or protected within the fibrils, have been reported for other proteins previously (74,75).…”

Exclusion of the Native α-Helix from the Amyloid Fibrils of a Mixed α/β Protein

“…In common with two previous studies of amyloid fibrils, 31,43 the H/D exchange kinetics of cystatin fibrils do not report on local differences in hydrogen bond stability for protected amide protons. A priori, it is possible that hydrogen bonds within the fibril are broken and reformed without greatly altering its overall structure.…”

“…In the case of β 2 -microglobulin 12,30 and transthyretin, 13 six of the eight β-strands of the native structures are protected within the amyloid fibrils, while the N and C-terminal strands are unprotected. In contrast, cold shock protein A 43 and an isolated SH3 domain 31 have protected regions that do not correlate with the secondary structure of the globular proteins. None of these proteins shows evidence of 3D domain swapping in the pattern of protection within the fibril.…”

High sensitivity 1H-NMR spectroscopy of homeopathic remedies made in water

“…While it could be argued that even 10% DMSO could disrupt H-bonding and thereby denature (i.e. destroy) H-bond structure [35], this cannot be said about D 2 O. Nor do we believe that the addition of the marker could have interfered with the detection of structuring.…”