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, protein 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 continued using 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 design appropriate inhibitory drugs that act on ternary structure rather than active site, thus avoiding VD(J) host disruption. We revisited A-tract bending studies of DNA (ms resubmitted) and continued using nucleotide analogs to reveal local disruptions in DNA structure (e.g., base flipping). We have continued and expanded our 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, and finding only a 1.5ps """"""""bulk water """""""" response. More important, we have also reexamined the work done by others on proteins such as Monellin, finding it is subject to QSSQ. Others interpreted the spectral shift as a property of the whole system, caused by water desorbing from protein, when in fact the selective loss on one side of a spectrum by QSSQ leads to a false shift in this range. We have submitted a manuscript to clarify that local quenching is the dominant mechanism, calling the desorbtion model into question. We published collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and we had quantified these reservoirs during changes in redox state and compartmental concentration. We are extending these studies in our new 2-photon fluorescence lifetime microscopy facility (collaboration with Microscopy Core) and obtaining images of NADH binding levels in the mitochondria of intact isolated cardiac myocytes. Recently, we have been carefully revisiting these measurements with NADH loaded vesicles to quantify aggregation of fluorophores and potential homotransfer- effects that might distort the free/bound ratio under extreme subcellular conditions. We assembled a 2-photon Fluorescence (Cross) Correlation Spectrometer with imaging capabilities and tested it on small molecules and on fluorescent proteins in transfected cells. In particular, nuclear transporter proteins were found to have significantly different mobilities (ability to diffuse laterally) in cytoplasm vs. nucleus. We began examining integrase assembly on LTR DNA with the same system. FCCS is a useful tool for quantifying mobility and stoichiometry of sparse proteins either in solution or in a cell.
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