Areas of Interest
It is widely recognized that to be biologically functional proteins have to be folded into specific structures and that, therefore, loss of structure leads to loss of function. Recent studies have demonstrated that in some cases a protein's structural alterations not only lead to loss of function, but can actually convert the protein into a cytotoxic form. This process features in a number of devastating human diseases including Alzheimer's disease, Parkinson's disease, mad cow disease and type-2 diabetes, to mention a few. In all these diseases the native structure of a specific protein or peptide is altered into a new structure that aggregates to form oligomeric species that bind to, and damage, cell membranes leading to cell death. Current research in our laboratory aims to characterize the mechanisms underlying this behavior and identify the molecular entities that feature in cellular toxicity. We focus mainly on the amyloid beta peptide, associated with Alzheimer's disease, and the islet amyloid peptide associated with type-2 diabetes. We are working on characterizing the structural alterations and molecular interactions that lead to the development of membrane-bound aggregates by these proteins, and on the mechanism by which these species induce cell death. To gain basic mechanistic understanding we use phospholipid liposomes as models for cell membranes, while to study the cytotoxicity we employ cultured neuronal-type cells.
Our elucidation of the structural evolution and membrane interactions of these proteins is accomplished using a variety of molecular-biological, biochemical and biophysical approaches. Laser-based optical spectroscopic techniques, and in particular time-resolved fluorescence, Forster resonance energy transfer circular dichroism and light scattering are used to follow protein conformational changes and aggregation in real time and serve in the development and testing of strategies for the inhibition of toxicity. Of special importance to our studies is the application of single molecule spectroscopy. This technique allows us to work with very low protein concentrations, as found in vivo, and to obtain unprecedented resolving power by following the interactions of individual peptide oligomers. Single molecule spectroscopy thus allows us to address mechanistic details of the origin and evolution of cytotoxicity at a level of detail that is impossible to achieve by conventional experimental approaches.
Honors & Awards
2001 Distinguished Director Award
2002 Ellison Medical Foundation Senior Scholar Program
Shi, J, Dertouzos,J, Gafni, A, Steel, DG, Palfey, BA: Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis. Proc. Nat. Acad. Sci. USA 2006; 103 5775-5780. PMID: 16585513
Shi, J, Gafni, A and Steel, DG: Simulated Data Sets for Single Molecule Kinetics: Some Limitations and Complications of Data Analysis. Eur. Biophys. J. 2006; 633-645. PMID: 16676175
Pattaramanon, N, Sangha, N and Gafni, A: The carboxy-terminal domain of HSF1 is intrinsically unstructured and can be induced to fold. Biochemistry 2007; 46, 3405-3415.
Gafni A and Walter, N.: The interdisciplinary biophysics graduate program at the University of Michigan. Biopolymers 2008, 89, 256-261. PMID: 18293398
Brender, JR, Lee, EL, Cavitt, MA, Gafni, A, Steel, DG and Ramamoorthy, A: Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide. J. Am. Chem. Soc. 2008: 130, 6424-6429. PMID: 18444645
Shi, J., Gafni, A. and Steel, DG: Application of single molecule spectroscopy in studies of enzyme kinetics and mechanisms. Meth. Enzymol. 2008, in press.
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