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, which results in the formation of insoluble fibrils. H-1-N-15 HSQC, 3D HSQC-NOESY, 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 observed during the entire course of the reaction. Under lag phase conditions, N-15 resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration. The dependence of N-15 T2 values on spin echo period duration demonstrates that line broadening is due to fast NMR exchange between acid-denatured monomers and soluble aggregates. Exchange contributions to T2 relaxation correlate with the squares of the chemical shift differences between native and acid-denatured CspA, and point to a stabilization of native-like structure upon aggregation Time-dependent changes in N-15 T2 relaxation accompanying the exponential phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggregate growth. CspA serves as a useful model system for exploring the conformational determinants of denatured protein misassembly. (C) 1999 Academic Press.