title: An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils.
abstract: At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils. 1H-15N HSQC, 3D HSQC-NOESY, and 15N 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 1H-15N HSQC cross-peaks, corresponding to acid-denatured monomers, is observed during the entire course of the reaction. Under lag phase conditions, 15N resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration. The dependence of 15N 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 15N 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.
authors.affiliation: Department of Structural Biology, University of Basel, Switzerland. alexandrescu@ubaclu.unibas.ch
authors.forename: A T
authors.initials: AT
authors.lastname: Alexandrescu
authors.forename: K
authors.initials: K
authors.lastname: Rathgeb-Szabo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D002352:Carrier Proteins / Q000737:chemistry* / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011108:Polymers; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D011108:Polymers