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 pursued protein-protein association within living cells, ultrafast protein solvation, mitochondrial energetics and ultrafast microscopy development. ? We also continued studies of DNA using fluorescent nucleotide analogs that reveal disruptions in DNA structures. We published a method for SNP detection using an enzyme that clips apart two differently colored probes that interact unless a sequence match is made.? We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early """"""""quasistatic self-quenching"""""""" processes. We found extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, GB1,etc.), as they detect previously silent conformers engaged in ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ that others attributed to a class of unique water molecules that desorb from protein in 20ps. We demonstrated (with several other proteins this year) that local quenching is the dominant mechanism in all but a locally unstructured protein. We published a molecular dynamics study of the ultrafast """"""""libration"""""""" (oscillatory, restricted motion) that flat fluorescent hydrocarbons do in solvents, rationalizing the subpicosecond polarization data we previously collected and solving a controversy about the process of emitting polarized light.? 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 in collaboration with Microscopy Core and LCE, we are refining 'Decay-Associated Images' of NADH binding within isolated cardiac myocytes. This year, we continued CARS (Coherent AntiStokes Raman Spectroscopy) microscope engineering to permit us to uniquely image the nonfluorescent oxidized form of NADH, NAD+ ( and very recently, cell water itself).? We have used our 2-photon FCS (Fluorescence (Cross) Correlation Spectrometer) with imaging capabilities to study the transport and binding of fluorescent proteins in both stably and transiently transfected cells. In vitro, we studied integrase assembly on HIV-LTR DNA and the integration host factor/HU binding of DNA. 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 downregulating interactions of the HIV nef protein with important cell-surface markers like CD4 and HLAA2 (organelle dependent). We also employed FCS to study the mobility of very low levels of C-myc ,finding exogenous C-myc is much more mobile than native protein.? We built (and pursued patents on) light collection devices for multiphoton microscopy of tissue that salvage any light that is emitted by the sample but does not enter the pupil of the objective. We showed that deep in rat brain slices, GFP labeled myosin gave >7X brighter signals using our TED(Total Emission Detection (=""""""""Morelight"""""""") ) device. A manuscript was published and we designed a new version for in vivo multiphoton imaging. We began collaborative simulation of deep multiphoton imaging with NICHD to seek the theoretical limits of such devices, with or without phase compensation array devices, and a prototype Adaptive Optics microscopy testbed is under construction.
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