The triplet state of tryptophan residues in proteins decays on the msec-sec time scale and is highly sensitive to the microenvironment of this residue. Long-lived room temperature phosphorescence (RTP) is invariably observed from tryptophan residues buried in the rigid, hydrophobic cores of globular proteins and can therefore be used to specifically probe aspects of these domains, including dynamics of interactions that occur on the time scale of the excited triplet state or longer. In his previous work, several triplet- state-based methodologies have been developed, demonstrated, and utilized to determine distances on the molecular level, to detect folding and unfolding protein intermediates with increased resolution, to observe heterogeneity of proteins in solution and to follow the slow annealing of a protein core domain during refolding. Dr. Gafni has strong indications from his work, as well as that of others, that this annealing may be a general phenomenon in proteins and is likely to cause the conformational modifications observed in proteins in cells of old animals. The long-term objective of this proposal is to apply triplet state-based methodologies to problems of structure, stability, and slow dynamics of proteins (such as the conformational changes that occur during folding or annealing of the protein core). The major research effort to be pursued in this proposed work will focus on the folding and stabilization of E. coli alkaline phosphatase in vitro as well as in vivo. The in vivo work is based on the exceptionally long-lived triplet state of this enzyme which enables its resolution from background luminescence from the cell. The research effort will place special emphasis on the role of individual amino acid residues in stabilizing the folded state and on the protein annealing mentioned above. To this end, he will prepare a number of AP mutants in which amino acid residues in the immediate environment of the phosphorescent tryptophan will be replaced by smaller residues. The structures of the mutants will be determined by x-ray crystallography, and the size of cavities formed by each mutation will be determined and correlated with protein stability, with several RTP parameters (lifetime, width of lifetime distribution, rate of D/H exchange) and with the rate of protein annealing. The latter variables are expected to trace the increased lability of the core due to cavity formation, as does the stability. The second research direction will focus on the relationship between changes in RTP decay and changes in structural details of proteins. In most cases, these changes are not observable by other means (e.g., UV-CD). Hence, Dr. Gafni proposes to determine the electronic energy transfer rate from the Trp triplet state via the exchange mechanism and measured by sensitized luminescence from Tb3+ bound to the metal binding sites of AP. This approach, which depends exponentially on the distance between Trp and Tb3+, has been demonstrated by Dr. Gafni and has potentially greater sensitivity over other methodologies.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG009761-08
Application #
2607651
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Finkelstein, David B
Project Start
1990-09-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1999-11-30
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Other Health Professions
Type
Schools of Medicine
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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