An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils

At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, whic h results in the formation of insoluble fibrils. H-1-N-15 HSQC, 3D HSQC-NOE SY, and N-15 T2 NMR experiments have been used to characterize the soluble components of this reaction. The kinetics of self-assembly show a lag phase followed by an exponential increase in polymerization. A single set of H-1 -N-15 HSQC cross-peaks, corresponding to acid-denatured monomers, is observ ed during the entire course of the reaction. Under lag phase conditions, N- 15 resonances of residues that constitute the beta-strands of native CspA a re selectively broadened with increasing protein concentration. The depende nce of N-15 T2 values on spin echo period duration demonstrates that line b roadening is due to fast NMR exchange between acid-denatured monomers and s oluble aggregates. Exchange contributions to T2 relaxation correlate with t he squares of the chemical shift differences between native and acid-denatu red CspA, and point to a stabilization of native-like structure upon aggreg ation Time-dependent changes in N-15 T2 relaxation accompanying the exponen tial phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggreg ate growth. CspA serves as a useful model system for exploring the conforma tional determinants of denatured protein misassembly. (C) 1999 Academic Pre ss.