Temperature jump and fast photochemical oxidation probe submillisecond protein folding.
We report a new mass-spectrometry-based approach for studying protein-folding dynamics on the submillisecond time scale. The strategy couples a temperature jump with fast photochemical oxidation of proteins (FPOP), whereby folding/unfolding is followed by changes in oxidative modifications by OH radical reactions. Using a flow system containing the protein barstar as a model, we altered the protein's equilibrium conformation by applying the temperature jump and demonstrated that its reactivity with OH free radicals serves as a reporter of the conformational change. Furthermore, we found that the time-dependent increase in mass resulting from free-radical oxidation is a measure of the rate constant for the transition from the unfolded to the first intermediate state. This advance offers the promise that, when extended with mass-spectrometry-based proteomic analysis, the sites and kinetics of folding/unfolding can also be followed on the submillisecond time scale.
Department of Chemistry, Washington University, One Brookings Drive, Saint Louis, Missouri 63130, United States.
Jiawei
J
Chen
Don L
DL
Rempel
Michael L
ML
Gross
D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D010777:Photochemistry; D017510:Protein Folding; D040901:Proteomics / Q000379:methods*; D013696:Temperature; D013997:Time Factors