Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure, assembly and flexibility of complex macromolecules. This year, we continued collaborative studies into DNA-protein interactions. We remain interested in the oligomerization and DNA binding of HIV-integrase, the enzyme used by the AIDS virus to incorporate itself into human DNA. We employed FRET with single-tryptophan mutants to measure site-specific distances between Trp and the end of the viral DNA, including kinetic measurement during 3' processing.. We also tested the ability of pyrene maleimide-labeled integrase mutants to form *excimers*, transient (NS) crosslinks in the excited state that create a green fluorescence where only violet was present without them. These excimers provide a measure of oligomerization useful in affirming the assembly of tetramers required for strand transfer. We prepared solubility-enhancing mutations for this difficult enzyme, and we used ultracentrifugation to quantify DNA -induced assembly in a salt-dependent fashion. We have continued preparation of labeled single-cysteine versions for FRET and excimers. Our scheme is to build a """"""""scaffold"""""""" of distances that define the complex, to help drug design. We also completed and published studies of the ~400-femtosecond librations of platelike molecules (perylene and tetracene, with sizes similar to tryptophan) inside solvent """"""""pockets"""""""" to prepare for similar studies in proteins. Measurement of this libration settled a longstanding controversy about anisotropy origins below 0.40 (the """"""""ro defect""""""""). We have completed molecular dynamics and quantum mechanical simulations with Drs. Brooks and Wu that confirm the libration, and not internal oscillator displacement, causes our transient term. We have continued femtosecond upconversion studies of peptides to quantify early (possibly electron ejection) events (leading to solvated electrons) that explain the """"""""quasistatic self-quenching"""""""" we had previously seen in peptides and proteins. We began studies of protein *solvation* on the 330fs-200ps time scale, using mutants of IIAGlc protein. We continued collaborative studies with LCE into the status of a primary fuel of isolated heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and we have quantified these reservoirs during changes in redox state and compartmental concentration, recently leading to a model for the affinity of relevant nicotinamide binding sites.
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