An argon laser will be used in fluorescence decay studies of creatine kinase, cascade polymers, nitrate reductase and an investigation of the static quenching limit. Intrinsic and extrinsic fluorescence lifetime and anisotropy investigations of creatine kinase folding and conformation are proposed. A comprehensive set of equilibrium and kinetic experiments, including quenching studies are proposed to describe intermediates in the folding pathway and the temporal relationship between folding and association of subunits. Experiments will assess the internal environment of cascade polymers in relation to synthetic micelles, using lipophilic probe lifetimes and anisotropies. Experiments are proposed to evaluate the solvent characteristics of the internal domain and the degree of packing using diphenylhexatriene, a well- characterized probe of micelle dynamics. A study is presented to characterize the relationship between catalytic properties and the conformations of the dimer, tetramer and isolated domains of nitrate reductase. Experiments are proposed to examine the assembly of the tetrameric state, using the intrinsic fluorescence and thiol-labelled protein as reporters of conformational changes in the presence of effectors of activity. Lifetime measurements will be used to obtain quenching constants for perylene and 9,10 dicyanoanthracence by carbon tetrachloride and used in calculation of the static quenching limit. The argon laser is a high intensity excitation source required for examining intrinsic fluorescence lifetimes and extrinsic fluorophore anisotropies. The multiple excitation wavelengths will be useful in global analysis of overdetermined data sets to characterize decay laws using discrete and distributed models.

Agency
National Science Foundation (NSF)
Institute
Division of Biological Infrastructure (DBI)
Type
Standard Grant (Standard)
Application #
9120260
Program Officer
Michael K. Lamvik
Project Start
Project End
Budget Start
1992-07-01
Budget End
1993-12-31
Support Year
Fiscal Year
1991
Total Cost
$41,240
Indirect Cost
Name
University of South Florida
Department
Type
DUNS #
City
Tampa
State
FL
Country
United States
Zip Code
33612