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, solvation, and energetics. 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 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 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 A-tract bending studies of DNA (ms to be submitted), began studies of the orientation of benzopyrene adducts on stepped DNA helices undergoing repair, and published studies of unique hybridization probes for DNA /RNA amplification and detection. We continued 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. We performed molecular dynamics and quantum mechanical simulations with Drs. Brooks and Wu that confirm the libration is a plausible source of the sub-ps transient term. We have completed femtosecond upconversion studies of peptides to quantify early (possibly electron ejection) events (leading to solvated electrons) that explain the QSSQ """"""""quasistatic self-quenching"""""""" we had previously seen in peptides and proteins. We found extremely rapid (10-100ps) nonradiative events that are also important in protein studies, as they imply conformers with facile charge transfer. We continued studies of protein *solvation* on the 330fs-200ps time scale, using mutants of IIAGlc protein. More important, we have also reexamined the work done by others on proteins such as Monellin, testing it for any QSSQ in this range. 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 and a manuscript submitted. We extended these studies by building a 2-photon fluorescence lifetime microscopy facility in the core and obtaining images of NADH binding levels in the mitochondria of intact isolated cardiac myocytes.
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