An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.
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 (approximately 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 approximately 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.
Alexandrescu
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AT
Department of Molecular and Cell Biology, University of Connecticut, 75 N. Eagleville Rd. U-3125, Storrs, CT 06269-3125, USA.
D000682:Amyloid / Q000737:chemistry*; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry*; D004268:DNA-Binding Proteins; D003903:Deuterium; D006360:Heat-Shock Proteins; D006801:Humans; D006859:Hydrogen; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011487:Protein Conformation
D000682:Amyloid; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; C094867:YBX3 protein, human; D006859:Hydrogen; D003903:Deuterium