Time-resolved room temperature tryptophan phosphorescence in proteins.

The application of luminescence, primarily fluorescence, to the study of protein structure and dynamics has been extensively exploited to facilitate the understanding of complex biological problems. The interest in the application of phosphorescence, however, shows that new and complementary information can be had by careful optical studies of the phosphorescence lifetime. As in the early days of fluorescence spectroscopy in proteins, a complete and rigorous interpretation of the room temperature phosphorescence remains to be developed; nevertheless, it is clear that time-resolved phosphorescence yields new information on proteins in solution, for example, the detection of subtle conformational changes during protein folding, which is outside the sensitivity of earlier techniques. In addition, the great sensitivity of the phosphorescence lifetime to structural changes associated with rigidity and of nearby quenchers suggests that detailed structural information can be obtained when this approach is combined with the power of site-directed mutagenesis or other more biophysical techniques such as energy transfer to attached acceptors. We have presented basic aspects of time-resolved room temperature phosphorescence spectroscopy and demonstrated some useful features of the spectroscopic signals as well as the general approach to data analysis. However, it should be understood that extensions of this approach will easily allow faster and improved time resolution with greater sensitivity to highly quenched phosphorescing states. In addition, many extensions of this approach that are common to fluorescence spectroscopy have yet to be developed. For example, combining time-resolved phosphorescence with anaerobic stopped-flow techniques and more rapid data acquisition electronics will enable studies of conformational dynamics with considerably shortened dead times. Other possibilities include extending the preliminary studies of in vivo-based spectroscopy, such as to microscopy. In conclusion, time-resolved phosphorescence presents a new dimension to biophysical methodologies for the study of proteins, and it is likely that this area will continue to grow in capability as the fundamental understanding improves.