Ari Gafni, Ph.D. | Institute of Gerontology

Research Professor, IoG
Research Professor, Biophysics Research Division
Professor of Biological Chemistry
Medical School

300 North Ingalls
Ann Arbor, MI 48109-2007
Tel: (734) 936-2156
Fax: (734) 936-2116
Email: arigafni@umich.edu

The main research interest in our laboratory focuses on protein folding mechanisms and on how misfolded proteins feature in aging and in age-associated diseases. While the concept that the three dimensional structure of a protein is encoded in its amino acids sequence is a central tenet of biology, how this information is utilized in directing the polypeptide chain to fold to a unique, biologically active, structure is unclear. This problem, often termed "the second half of the genetic code," is of great importance as its solution will allow us to predict structures of proteins from their amino acids sequences, and to engineer more stable, longer lived, proteins by introducing appropriate mutations. This understanding will open the door for engineering proteins with new structures and functions.

All living cells possess a group of special proteins, collectively called heat shock proteins (HSPs), that protect existing cellular proteins from becoming damaged and assist newly synthesized proteins to fold correctly. A prominent heat shock protein is HSP70 whose production in young cells is elevated many fold in response to a variety of stresses (elevated temperatures, toxic substances, ionizing radiation, etc.). Cells of senescent organisms show a marked decline in their ability to elevate the expression of HSP70 and, therefore, possess a much-reduced ability to cope with external challenges. Previous research has revealed that the age-related reduction in HSP70 production correlates with a decreased fidelity of binding of the HSP70 transcription factor (called HSF1) to a specific region on the DNA, the heat shock element (HSE). However, the molecular origin of this defect is not known. Work in this area has been hindered by the low abundance of HSF1 in mammalian cells (from which the old form of the protein is extracted) as well as by the structural and functional heterogeneity of HSF1 molecules in old tissues. To overcome these difficulties we are using novel methodologies based on single molecule detection. Single molecule spectroscopy allows us to work with minute amounts of material (tens to hundreds of molecules), and to discern mechanistic aspects which, when using conventional approaches, are masked by ensemble averaging. Our work focuses on identifying the molecular origin of the age related decline in the functional fidelity of HSF1. To this end we are currently working to characterize the structural changes responsible for HSF1 activation and DNA binding following heat sensing, and to identify the age-related alterations in HSF1 and how these impair its function. We expect that this research will provide a molecular explanation for how protein aging negatively impacts transcriptional activation of target genes.

A related current research effort in our laboratory addresses mechanistic aspects of the tissue-specific production of amyloid deposits, the hallmark of a number of devastating age-associated human diseases including Alzheimer's disease and non-insulin-dependent diabetes. While the proteins that form amyloid deposits in each of these diseases share no sequence homology or tertiary-structure similarity, their aggregation products are morphologically remarkably similar and at the molecular level they all possess a common structural motif. The research focuses on the highly specific molecular interactions that lead to amyloid formation, and on the forces that stabilize this structure with an effort to identify the reasons for the strong age-relatedness of this deposition and to develop strategies for its inhibition. Our most recent work addresses the potential cytotoxicity of small molecular aggregates produced by amyloid-forming peptides, that appear early during amyloid formation and also serve as seeds to facilitate the process.

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Recent Publications

Subramaniam, V., Steel, D.G., and Gafni, A. (2001) Room temperature tryptophan phosphorescence as a probe of structural and dynamic properties of proteins. In "Topics in fluorescence spectroscopy", Vol. 6 (J. Lakowicz, Ed.) pp 52-72.

Fischer, C. J., Schauerte, J. A., Wisser, K. C., Steel. D. G., and Gafni, A. (2001) Differences in the pathways for unfolding and hydrogen exchange among mutants of E. coli alkaline phosphatase. Biochim. Biophys. Acta, 1545: 96-103.

Gafni, A. (2001) Protein Structure and Turnover, in Handbook of the Biology of Aging, 5th Edition (E. Masoro & S. Austad, Editors) Academic Press; pp 59-83.

Dirnbach, E., Steel, D., and Gafni, A. (2001) Magnesium binding to alkaline phosphatase correlates with slow structural changes during folding, Biochemistry, 40: 11219-11226.

Fischer C.J., Gafni A., Steel D.G., Schauerte J.A. (2002) The triplet-state lifetime of indole in aqueous and viscous environments: Significance to the interpretation of room temperature phosphorescence in proteins. Journal of the American Chemical Society, 124: 10359-10366.

Rhoades, E. and Gafni, A. (2003) Micelle formation by a fragment of human islet amyloid polypeptide. Biophys. J, 84: 3480-3487.

Lewis MK, Wolanin P, Gafni A, and Steel DG. (2003) Near field scanning optical microscopy of single molecules by femtosecond two-photon excitation. In “Selected Papers on Multiphoton Excitation Microscopy” (Masters BR, ed) SPIE Milestone Series Vol. 175, pp 472-474.

Brender JR, Dertouzos JM, Shi J, Burmeister E, Palfey B, Ballou DP, Massey V, Gafni A, Steel D.G. (2003) Conformational regulation and heterogeneity of para-hydroxybenzoate hydroxylase studied at the single molecule level. Biophys. J. 84 (2): 124A-124A.

Shi J, Palfey BA, Dertouzos J, Jensen KF, Gafni A. and Steel DG. (2004) Single molecule study on kinetics and mechanism of dihydroorotate dehydrogenase (DHOD). J. Am. Chem. Soc. 126(22): 6914-6922.

Wisser K.C., Schauerte J.A., Burke D.T., Galecki A.J., Chen S., Miller R.A., Gafni A. (2004) Mapping Tissue-Specific Genes Correlated with Age-Dependent Changes in Protein Stability and Function. Arch. Biochem. Biophys., in press.

Sherman WA, Blouse GE, Perron MJ, Tran T, Shore JD, and Gafni A. (2004) Enthalpy measurement using calorimetry shows a significant difference in potential energy between the active and latent conformations of PAI-1. Biol. Chem, in press.