WW Domain Folding Complexity Revealed by InfraredSpectroscopy

摘要

Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-typeWW domain and tryptophan mutants exhibit single-exponential kineticswith a lifetime that corresponds to the slowest phase observed byinfrared spectroscopy. Mutant sequences provide evidence of an intermediatedry molten globule state. The slowest step in the folding of thisWW domain is the tight packing of the side chains in the transitionfrom the dry molten globule intermediate to the native structure.This study demonstrates that using multiple complementary probes enhancesthe interpretation of protein folding dynamics.