AN NMR-BASED QUENCHED HYDROGEN EXCHANGE INVESTIGATION OF MODEL AMYLOID FIBRILS FORMED BY COLD SHOCK PROTEIN A

摘要

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 D_2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D_2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D_2O (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.