The fluorescence of a protein is determined by interactions of the fluorophore with the protein matrix and with solvent. The usefulness of fluorescence spectroscopy is predicated on the ability to interpret the fluorescence signal in terms of specific molecular interactions. Although it is commonly held that we understand how such interactions influence fluorescence, this is not substantiated by available data. This not surprising given (i) the complexity of the environs of fluorophores in proteins; (ii) our minimal understanding of the dynamics of the protein matrix and of water interacting with the understanding of the dynamics of the protein matrix and of water interacting with the protein matrix; (iii) the lack of adequate theory describing the excited state of fluorophores, (iv) the lack of information on the effects of electrostatic interactions on fluorophore photophysics, and (v) the uncertainty (engendered by i-iv) of being able to devise good physical models to explain the experimental data. Any physical model must be able to explain all of the fluoresence properties of a protein (ie. quantum yield, emission spectra, r(t), and fluorescence quenching). Our approach to correlating protein structure and intrinsic (trypotophan) luminescence requires concurrent use of (a) steadystate and time (picosecond) resolved fluorescence spectroscopy (and other spectroscopic methods where necessary) to determine the fluorescence properties of proteins of known crystal structure, each containing a single trp residue but exhibiting different fluorescence properties; (b) Molecular dynamics simulations - including activated molecular dynamics calculations - to assess trp side chain motion, water accessibility and dynamics, and electrostatic interactions with the trp residue; (c) molecular graphics depictions; (d) Semi-empirical calculations on the photophysics of indole and other commonly used fluorophores in an attempt to provide at least a semiquantitative assessment of environmental effects on fluorescence. The principal initial goal is to examine further the views that interpretation of fluorescence lifetimes in terms of distribution of states is valid and that whereas emission spectrum reflects the effects of a static field in the trp environs. Lastly, we suggest that dipolar relaxation in proteins probably arises from re-orientation of solvent molecules adsorbed to the protein matrix adjacent to the fluorophore.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034847-08
Application #
3286567
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1985-08-01
Project End
1993-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Kamlekar, Ravi Kanth; Simanshu, Dhirendra K; Gao, Yong-guang et al. (2013) The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids. Biochim Biophys Acta 1831:417-27
Kenoth, Roopa; Kamlekar, Ravi Kanth; Simanshu, Dhirendra K et al. (2011) Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity? Biochemistry 50:5163-71
Kamlekar, Ravi Kanth; Gao, Yongguang; Kenoth, Roopa et al. (2010) Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold. Biophys J 99:2626-35
Kenoth, Roopa; Simanshu, Dhirendra K; Kamlekar, Ravi Kanth et al. (2010) Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold. J Biol Chem 285:13066-78
Kirk, William (2008) Solvent Stokes'shifts revisited: application and comparison of Thompson-Schweizer-Chandler-Song-Marcus theories with Ooshika-Bakshiev-Lippert theories. J Phys Chem A 112:13609-21
Kirk, William; Klimtchuk, Elena (2007) Photophysics of ANS. III: Circular dichroism of ANS and anilinonaphthalene in I-FABP. Biophys Chem 125:24-31
Kirk, William (2007) Photophysics of ANS. II: Charge transfer character of near-UV absorption and consequences for ANS spectroscopy. Biophys Chem 125:13-23
Kirk, William; Kurian, Elizabeth; Wessels, William (2007) Photophysics of ANS. V. Decay modes of ANS in proteins: the IFABP-ANS complex. Biophys Chem 125:50-8
Klimtchuk, Elena; Venyaminov, Sergei; Kurian, Elizabeth et al. (2007) Photophysics of ANS. I. Protein-ANS complexes: Intestinal fatty acid binding protein and single-trp mutants. Biophys Chem 125:1-12
Kirk, William; Wessels, William (2007) Photophysics of ANS. IV. Electron transfer quenching of ANS in alcoholic solvents and mixtures. Biophys Chem 125:32-49

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