Nanosecond time-resolved and steady-state fluorescence techniques will be used to investigate the static and dynamic structures of proteins and macromolecular assemblies. Both intrinsic fluorophores and extrinsic fluorescence probes will be utilized. Procedures will be devised to differentiate complex decay having its origin in ground-state microheterogeneity from that which has its origin in excited-state interactions. The work will emphasize the use of """"""""overdeterminations"""""""" with the development of analysis algorithms which combine the analysis of fluorescence decay data obtained at different wavelengths, temperature, pH and at various quencher concentrations. Efforts will be focused on the fluorescence of tryptophan and extrinsic probes such as substituted naphthalene derivatives and other fluorophores of interest. Fluorescence spectroscopy will be used to study the interactions of Enzyme I, HPr and III/Glc, three proteins of the phosphoenolpyruvate:glycose phosphotransferase system. Intrinsic fluorescence and resonance energy transfer, as measured by nanosecond fluorometry will be used to study the folding of Staphylococcal nuclease. The energy transfer donor will be the single tryptophan residue and the acceptor will be conjugated to single cysteine residues introduced at unique sites in the protein. Fluorescence studies will be carried out on the structure of horse liver alcohol dehydrogenase and thioredoxin. Excited-state solvent relaxation will be investigated in order to better understand how fluorescent probes sense changes in their environment. The focus of the research is to develop new ways to use steady-state and nanosecond time-resolved fluorescence to uncover points of ignorance regarding biological macromolecules and macro-assemblies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM011632-27
Application #
3268283
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1977-09-01
Project End
1992-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
27
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Brown, M P; Shaikh, N; Brenowitz, M et al. (1994) The allosteric interaction between D-galactose and the Escherichia coli galactose repressor protein. J Biol Chem 269:12600-5
Wu, P; Li, Y K; Talalay, P et al. (1994) Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism. Biochemistry 33:7415-22
Wu, P; Brand, L (1994) Resonance energy transfer: methods and applications. Anal Biochem 218:1-13
Chauvin, F; Brand, L; Roseman, S (1994) Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer transition kinetics by fluorescence anisotropy. J Biol Chem 269:20270-4
Wu, P; Brand, L (1994) Conformational flexibility in a staphylococcal nuclease mutant K45C from time-resolved resonance energy transfer measurements. Biochemistry 33:10457-62
Chauvin, F; Brand, L; Roseman, S (1994) Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer equilibrium by fluorescence anisotropy. J Biol Chem 269:20263-9
Hirshfield, K M; Toptygin, D; Packard, B S et al. (1993) Dynamic fluorescence measurements of two-state systems: applications to calcium-chelating probes. Anal Biochem 209:209-18
Wu, P G; James, E; Brand, L (1993) Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements. Biophys Chem 48:123-33
Toptygin, D; Brand, L (1993) Fluorescence decay of DPH in lipid membranes: influence of the external refractive index. Biophys Chem 48:205-20
Rice, K G; Wu, P; Brand, L et al. (1993) Modification of oligosaccharide antenna flexibility induced by exoglycosidase trimming. Biochemistry 32:7264-70

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