STRUCTURAL FLUCTUATIONS IN PROTEINS REVEALED BY TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Time-resolved fluorescence spectroscopy permits the direct assessment of protein motions in the picosecond-nanosecond time-scale, i.e., in a time-window compatible with the observation of relevant motions of the protein matrix. The intrinsic fluorescence emission from tryptophan and tyrosine residues provides a convenient tool to follow these dynamic events in proteins. In the present investigation, the use of time-resolved fluorescence spectroscopy to monitor protein dynamics is illustrated by a study of the effects of temperature and calcium binding on the internal dynamics of the calcium-binding protein, parvalbumin, and by an investigation of the effects of hydration on the mobility of tryptophan side chains of lysozyme and azurin in low-water media. Our results showed that measurements of both fluorescence intensity and anisotropy decays provided complementary information regarding the flexibility of aromatic side chains in the proteins investigated, which could be correlated with environmental effects on protein structure.