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Measuring Alpha-Synuclein Conformational Dynamics Using FCS
Elizabeth Rhoades, Assistant Professor of Molecular Biophysics & Biochemistry, Yale University.
David Eliezer, Associate Professor of Biochemistry, Weill Medical College.
Funding: NIH/NIGMS 1R21GM084391 (5/08-4/10).

Alpha-synuclein (αS) is a major component of Lewy body deposits in the brain and a hallmark of Parkinson’s disease. Intrinsically unstructured in solution, it is known to adopt an α-helical structure upon binding to lipid membranes. Its native function is poorly understood, but it has been suggested that its interaction with lipid membranes may play a role both in its native function as well as in the pathology of Parkinson’s disease. In the disease state, αS oligomerizes into a fibril with a cross-β amyloid structure. In this project we use FCS to measure the binding of αS to large unilamellar vesicles (LUVs). We will also determine the long-range contacts made between different regions in αS. We have demonstrated the use of FCS as a tool for quantifying the binding of αS to LUVs of various lipid compositions (Rhoades et al 2006). The diffusion of fluorescently-labeled αS in a solution of LUVs can be fit to a model with two diffusing species, corresponding to LUV-bound and free αS. We have shown that αS binds preferentially to LUVs made from acidic lipids, and this can be reversed by increasing the concentration of salt, implicating an electrostatic interaction. Binding is also enhanced in the presence of less bulky lipid headgroups.

We plan to continue the successful collaboration with Professors Rhoades and Eliezer by using FCS to characterize the long-range contacts formed in αS. FCS will be used to measure the collisional quenching of a site-specific Alexa-488 label when it makes non-native contacts with certain amino acids elsewhere on the protein. We will screen mutants that have the fluorophore at different positions within the protein to determine the specific contacts. Since αS does not contain any Tryptophan (Trp) residues which are by far the strongest quencher of Alexa-488 fluorescence, we will also mutate in Trp residues at positions that are predicted to make long-range contacts with the region where the fluorophore is located.  This project benefits from advanced fluctuation analysis capabilities that are being developed.

References

Rhoades, E., T. F. Ramlall, W. W. Webb and D. Eliezer, 2006. "Quantitation of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy," Biophysical Journal 90(12), 4692-4700.

 

 

 



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