Optical spectroscopic techniques have had a major impact on the study of protein structure and mechanisms. However, only a small fraction of the work has employed triplet-state based spectroscopies. The proposed research will combine the skills and facilities of a biophysicist (the principal investigator) and a laser spectroscopist (co-principal investigator) to develop several laser-based methodologies which will form the basis of an extensive program to apply the triplet-state spectroscopies to important problems in molecular biophysics. A wealth of significant new information on protein structure and interactions in solutions is expected from this approach. The methodologies to be developed include: (a) A system from the rapid acquisition (based on a single excitation pulse) of phosphorescence decay kinetics allowing for real-time monitoring of structural transitions in proteins as involved in unfolding and refolding: (b) Use of diffusion-enhanced Forster-type energy transfer from intrinsic triplet-state donors to study protein structure and interactions though mapping of the distances of phosphorescent residues from the surface of the protein; (c) Time integrated and time resolved circularly polarized phosphorescence (CCP) to applied in detailed studies of protein conformations as well as conformational changes that occur on a time scale comparable to the triplet state lifetime; (d) Laser- based triplet-triplet absorption to be used to determine triplet state decay patterns in systems with low phosphorescence yield. An extension of this approach to include time-resolved circular dichroism of triplet- triplet transitions will provide information complementary to that derived from CPP. These approaches will be developed using model enzymes chosen by virtue of their stability, phosphorescence properties and structural information and will be applied to several significant problems in molecular biophysics. These include conformational isomerism in enzymes, structural variability among refolding intermediates, subunit association in oligomeric enzymes and enzyme-enzyme complex formation patterns. Another interesting application will be the study of the structural modifications responsible for the documented differences between enzymes purified from tissues of young and old animals.
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