We have constructed (and continue to develop) a laser-based facility for time-resolved fluorescence spectroscopy of biomolecules. This facility provides rapid collection and analysis of luminescence data related to macromolecular size, flexibility, folding and structural fluctuations. Our time-correlated laser fluorometer was used to study the folding and dynamics of several proteins. We focused most of our effort on DNA- binding proteins that control the transcription of DNA blueprints into the """"""""field copies"""""""" (m-rna) used to build proteins in cells. Fluorescence was used to measure distances between proteins and the sections of DNA they control, to look at the wobbling of proteins in the complex, and to reveal internal changes in the bound protein. We completed our studies of oct-pou, a transcription factor that recognizes two """"""""classes"""""""" of sites. It was seen to """"""""read"""""""" DNA by changing shape. We continued collaborative studies on HIV integrase, the enzyme responsible for incorporation of the AIDS virus into host cells; on Heat Shock Factor domains, to help ascertain its' binding mechanism; and on TFIIIA - internal control region (5s RNA gene) interactions. We began work this year on specially labeled GAL4(5HT) versions of the (herpes) activator VP-16 to test the flexibility in complexes with promoter elements like TBP. We also studied the self-associative properties of globin activator USF and interleukin 8. This year we also continued efforts to adapt our laser instruments to the imaging of tissues. In particular, we patented two electronic laser scanning devices that use discoloration to find BB-sized objects almost an inch inside tissue. We also developed a more precise (30 micron) imager that can provide slices of skin images up to 1/8"""""""" deep, then scan sideways.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL001452-11
Application #
3757630
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
1994
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Michelman-Ribeiro, Ariel; Mazza, Davide; Rosales, Tilman et al. (2009) Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys J 97:337-46
Rosales, Tilman; Xu, Jianhua; Wu, Xiongwu et al. (2008) Molecular dynamics simulations of perylene and tetracene librations: comparison with femtosecond upconversion data. J Phys Chem A 112:5593-7
Harvey, John J; Brant, Steven R; Knutson, Jay R et al. (2008) SNP analysis using CataCleave probes. J Clin Lab Anal 22:192-203
Rosales, Tilman; Georget, Virginie; Malide, Daniela et al. (2007) Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy. Eur Biophys J 36:153-61
Combs, Christian A; Smirnov, Aleksandr V; Riley, Jason D et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J Microsc 228:330-7
Xu, Jianhua; Toptygin, Dmitri; Graver, Karen J et al. (2006) Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc. J Am Chem Soc 128:1214-21
Augustyn, Katherine E; Wojtuszewski, Kristi; Hawkins, Mary E et al. (2006) Examination of the premelting transition of DNA A-tracts using a fluorescent adenosine analogue. Biochemistry 45:5039-47
Blinova, Ksenia; Carroll, Stefanie; Bose, Salil et al. (2005) Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions. Biochemistry 44:2585-94
He, Liusheng; Bradrick, Thomas D; Karpova, Tatiana S et al. (2003) Flow cytometric measurement of fluorescence (Forster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm. Cytometry A 53:39-54
Kim, Soon-Jong; Beard, William A; Harvey, John et al. (2003) Rapid segmental and subdomain motions of DNA polymerase beta. J Biol Chem 278:5072-81

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