Major research activities during this reporting period include: (1) ongoing development of an ultrafast time-resolved laser spectroscopy laboratory; (2) time-resolved x-ray crystallographic studies of structural dynamics in proteins; and (3) giving invited lectures at national and international symposia as well as serving on scientific committees. We are developing the laser-based technology required to pursue time-resolved spectroscopic studies of protein dynamics from 100 fs to milliseconds using wavelengths ranging from the uv to the infrared. Some of the opportunities afforded by ultrafast spectroscopic methods have been published recently in two papers appearing in the Proceedings of the National Academy of Science. Ultrafast time-resolved laser spectroscopy requires mastery of many skills that traditionally arise from extensive hands-on experience. To shift the focus away from the technological demands and onto the protein systems of interest, we are developing computerized controls to optimize and control an ultrafast laser spectrometer. This effort will significantly enhance the quality and reproducibility of time-resolved spectra and will provide much greater flexibility in terms of accessible wavelengths. The infrastructure for implementing these controls is now largely in place and cutting-edge spectroscopic measurements should become nearly routine in the coming year. A multinational collaboration has been cultivated to pursue time-resolved structural studies of photoactive yellow protein (PYP) and myoglobin (Mb) using the technique of time-resolved x-ray crystallography. To date, diffraction data have been collected at the ESRF in Grenoble, France on both PYP and Mb with time resolution as fast as 150 ps. Our preliminary analysis of the Mb diffraction data are quite promising. For example, the crystal structure of V68F Mb*CO, measured 150 ps after photolysis, reveals three significant features: displacement of the iron toward the proximal histidine, depletion of bound CO, and appearance of unbound CO in a docking site. These structural features are being compared with those for wild-type Mb*CO to explore the influence of this single point mutation on ligand rebinding dynamics, which, according to ultrafast time-resolved IR measurements, is significantly enhanced with this mutation. Analysis of the diffraction data is a lengthy process being pursued by collaborators, and additional results are expected in the coming few months.
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