Intrinsic Fluorescence Decay Mechanisms of Proteins is a multi- investigator, multidisciplinary project combining time-resolved fluorescence with NMR and molecular modeling. Our objective is to determine how time-resolved fluorescence of the three aromatic amino acids tryptophan, tyrosine, and phenylalanine can be used to obtain information about structure and conformational dynamics of proteins. This requires understanding how and to what extent ground-state and excited-state processes affect the fluorescence intensity decay kinetics. NMR is being used to help define important ground-state processes. Molecular modeling, based on X- ray crystal structures, is being used to compute properties of an aromatic amino acid's local environment for comparison with the conclusions obtained from fluorescence and NMR. In this initial grant period we are investigating well-defined, simple peptide and polypeptide analogues having a single aromatic amino acid by systematically substituting in each aromatic amino acid at the same position in the peptide chain. These studies on peptides and polypeptides are the basis for extending the investigation into several structurally related single-tyrosine proteins. The results of this interdisciplinary approach, emphasizing tyrosine fluorescence, will be used, through a continuation application, to extend this investigation to tryptophan fluorescence of proteins. This work will further our understanding of the time-resolved fluorescence of the aromatic residues, add significantly to our knowledge of the solution structure and conformational dynamics of proteins, and make time-resolved fluorescence techniques a more powerful tool for analyzing protein structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM039750-01
Application #
3296907
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1988-07-01
Project End
1992-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Rachofsky, E L; Laws, W R (2000) Kinetic models and data analysis methods for fluorescence anisotropy decay. Methods Enzymol 321:216-38
Zemsky, J; Rusinova, E; Nemerson, Y et al. (1999) Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor. Proteins 37:709-16
Bishop, S M; Ross, J B; Kohanski, R A (1999) Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft. Biochemistry 38:3079-89
Senear, D F; Ross, J B; Laue, T M (1998) Analysis of protein and DNA-mediated contributions to cooperative assembly of protein-DNA complexes. Methods 16:3-20
Bialik, C N; Wolf, B; Rachofsky, E L et al. (1998) Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay. Biophys J 75:2564-73
Ross, J B; Szabo, A G; Hogue, C W (1997) Enhancement of protein spectra with tryptophan analogs: fluorescence spectroscopy of protein-protein and protein-nucleic acid interactions. Methods Enzymol 278:151-90
Huang, Y T; Rusinova, E; Ross, J B et al. (1997) An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor. J Mol Biol 267:403-17
Rusinova, E; Ross, J B; Laue, T M et al. (1997) Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor. Biochemistry 36:12994-3003
Hermann, A; Laws, W R; Harpel, P C (1997) Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site. Protein Sci 6:2324-35
Driscoll, S L; Hawkins, M E; Balis, F M et al. (1997) Fluorescence properties of a new guanosine analog incorporated into small oligonucleotides. Biophys J 73:3277-86

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