Optical spectroscopic techniques have had a major impact on the study of protein structure and mechanisms. However, only a small fraction of the work has employed triplet-state based spectroscopies. The proposed research will combine the skills and facilities of a biophysicist (the principal investigator) and a laser spectroscopist (co-principal investigator) to develop several laser-based methodologies which will form the basis of an extensive program to apply the triplet-state spectroscopies to important problems in molecular biophysics. A wealth of significant new information on protein structure and interactions in solutions is expected from this approach. The methodologies to be developed include: (a) A system from the rapid acquisition (based on a single excitation pulse) of phosphorescence decay kinetics allowing for real-time monitoring of structural transitions in proteins as involved in unfolding and refolding: (b) Use of diffusion-enhanced Forster-type energy transfer from intrinsic triplet-state donors to study protein structure and interactions though mapping of the distances of phosphorescent residues from the surface of the protein; (c) Time integrated and time resolved circularly polarized phosphorescence (CCP) to applied in detailed studies of protein conformations as well as conformational changes that occur on a time scale comparable to the triplet state lifetime; (d) Laser- based triplet-triplet absorption to be used to determine triplet state decay patterns in systems with low phosphorescence yield. An extension of this approach to include time-resolved circular dichroism of triplet- triplet transitions will provide information complementary to that derived from CPP. These approaches will be developed using model enzymes chosen by virtue of their stability, phosphorescence properties and structural information and will be applied to several significant problems in molecular biophysics. These include conformational isomerism in enzymes, structural variability among refolding intermediates, subunit association in oligomeric enzymes and enzyme-enzyme complex formation patterns. Another interesting application will be the study of the structural modifications responsible for the documented differences between enzymes purified from tissues of young and old animals.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG009761-05
Application #
2051037
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1990-09-01
Project End
1995-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Other Health Professions
Type
Other Domestic Higher Education
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Fischer, Christopher J; Gafni, Ari; Steel, Duncan G et al. (2002) The triplet-state lifetime of indole in aqueous and viscous environments: significance to the interpretation of room temperature phosphorescence in proteins. J Am Chem Soc 124:10359-66
Dirnbach, E; Steel, D G; Gafni, A (2001) Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability. Biochemistry 40:11219-26
Fischer, C J; Schauerte, J A; Wisser, K C et al. (2001) Differences in the pathways for unfolding and hydrogen exchange among mutants of Escherichia coli alkaline phosphatase. Biochim Biophys Acta 1545:96-103
Fischer, C J; Schauerte, J A; Wisser, K C et al. (2000) Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence. Biochemistry 39:1455-61
Gershenson, A; Schauerte, J A; Giver, L et al. (2000) Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry 39:4658-65
Dirnbach, E; Steel, D G; Gafni, A (1999) Proline isomerization is unlikely to be the cause of slow annealing and reactivation during the folding of alkaline phosphatase. J Biol Chem 274:4532-6
Gershenson, A; Gafni, A; Steel, D (1998) Comparison of the time-resolved absorption and phosphorescence from the tryptophan triplet state in proteins in solution. Photochem Photobiol 67:391-8
Bergenhem, N C; Lee, S J; Gafni, A (1997) The stable, inactive but reactivatable, unfolding intermediate of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase differs from the age-modified form of this protein. J Gerontol A Biol Sci Med Sci 52:B240-4
Gafni, A (1997) Structural modifications of proteins during aging. J Am Geriatr Soc 45:871-80
Subramaniam, V; Steel, D G; Gafni, A (1996) In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state. Protein Sci 5:2089-94

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