Room temperature tryptophan phosphorescence spectroscopy as a probe of protein structure and dynamics.

Abstract

This thesis demonstrates the applicability of room temperature tryptophan phosphorescence to a variety of problems in protein structure and dynamics. Phosphorescence is able to measure many of the same quantities as fluorescence with better signal to noise ratio, and is uniquely capable of monitoring events in the msec-sec time scale. In our study of phosphorescence we developed a laser-based excitation system which provided us with a stronger signal to measure than that obtainable with flashlamp based systems, and the use of photon counting techniques in our detection system gave sensitive detection of signal as well as a digital format for easier and more accurate data analysis. A powerful application of room-temperature tryptophan phosphorescence was to the study of what is widely known as the protein-folding problem. Phosphorescence was used to demonstrate for the first time the presence of transient intermediate conformations of E. coli alkaline phosphatase during its unfolding in GdnHCl. We have also extended the theoretical basis of the use of energy transfer in the rapid diffusion limit, which requires long-lived donor emission such as phosphorescence. This thesis presents the energy transfer rate equation for a geometry which better represents real proteins, and also presents equations that show that in spite of the long decay time of the donor, steric effects can cause significant variations in transfer rate with variation in chromophore orientation angle. The theory developed was used in an experiment to directly measure the depth beneath the protein surface of the phosphorescent tryptophan in E. coli alkaline phosphatase, confirming the identification of this residue as Trp 109. These results demonstrate the utility of room temperature tryptophan phosphorescence protein studies. Although phosphorescence has the disadvantages of requiring sample deoxygenation and a more sensitive detection system, these are easily overcome by the methods described in this thesis and the references therein, making the benefits of studying room temperature tryptophan phosphorescence well worth the additional experimental steps required.