Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we studied DNA-protein interactions, ultrafast protein solvation, mitochondrial energetics and ultrafast microscopy. ? We are interested in the DNA binding of HIV-integrase, the enzyme used by the AIDS virus to stitch itself into human DNA. We prepared solubility mutations and continued preparation of fluorescently labeled versions for FRET and FCS studies of this complex equilibrium.? We also published A-tract dependent bending studies of DNA using fluorescent nucleotide analogs that reveal disruptions in DNA shape (e.g., base flipping).? We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early (possibly electron transfer) events that explain the QSSQ """"""""quasistatic self-quenching"""""""" often seen. We found extremely rapid (10-100ps) decays are important in protein studies, as they imply conformers with ultrafast charge transfer. We published a key study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, and finding it shows QSSQ. Others had interpreted (in a series of papers) the 20-picosecond spectral shift of Monellin as waters desorbing from protein, when, in fact, a fast decay process (QSSQ) created a false shift. We demonstrated that local quenching is the key mechanism, calling the desorbtion model into question.? We contined 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 previously quantified these reservoirs during changes in redox state. We are extending these studies in our new 2-photon fluorescence lifetime microscopy facility (collaboration with Microscopy Core) and obtaining Decay-Associated Images of NADH binding levels in the mitochondria of intact isolated cardiac myocytes. We have been carefully benchmarking these measurements with both NADH loaded and NADH/protein loaded vesicles to quantify aggregation of fluorophores and potential homotransfer- effects that might otherwise distort the free/bound ratio we recover under extreme subcellular conditions. ? We have used our 2-photon Fluorescence (Cross) Correlation Spectrometer with imaging capabilities? to study the transport and binding of fluorescent proteins in transfected cells. In particular, androgen receptor (nuclear) transporter proteins were found to have cross-correlation (proof of binding and cotransport) with the Tif2 cofactor in the nucleus. This level of interaction changed with effector drugs (ms. in press). We began examining integrase assembly (above) on HIV-LTR DNA? with the same system. FCCS is a useful tool for quantifying mobility and stoichiometry of dilute proteins either in solution or in a cell. The same system was used to study the aggregation of the HIV nef protein and (separately) Mu transposase.? We furthered our ultrafast microscopy collaborations by building a CARS microscope; testing on vesicles is underway.? We built (and filed for patents) a light collection device for multiphoton microscopy of tissue whose purpose is to salvage most of the light that is emitted by the sample but does not enter the pupil of the objective. We showed that in rat brain, GFP labeled proteins 120u deep were >4X brighter using our ?Morelight? device. Posters were presented and a manuscript is in preparation.
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