Biophysical Characterization of Alpha-Synuclein

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The long-term research objective of this project is to define how variations in amino acid sequence and surrounding environment of a- synuclein, the major protein component of Lewy bodies in Parkinson Disease affect structure, stability, self-association and/or aggregation, and fibril formation. The long-term goal of the career development plan in the academic setting is to become a fundamental researcher and scientific leader in the field of protein structure, stability, folding and aggregation. In particular, I will concentrate my efforts on the determinants (surrounding environment and amino acid sequence) of folding and misfolding of proteins and the relation of misfolding, aggregation, and precipitation to the neurological disorders which are so prevalent in the aged population. The urgency for biophysical research in this difficult area in which there are more questions than answers is very strong. By the mastering of new techniques, the fine- tuning of others with which I have already had experience, course work, technique workshops, conference presentations, and collaborative interactions throughout the course of the award, I will become complete as a scientist who is competent to address many of the questions and uncertainties which are inherent and continue to evolve in this field. The focus and general approach is to generate peptide models and expression systems for the production and purification of wild-type and variants bearing specific mutations of a-synuclein and (3-synuclein, a protein highly homologous to a-synuclein. In the first aim, the conformations of a-synuclein and (3-synuclein will be compared. The long-term goal of this aim is to determine the factors involved in dictating the native conformations of both a- and (3-synuclein. In the second aim, the hypothesis that (3-synuclein will not aggregate in vin-o will be tested. The long-term goal of this aim is to define the determinants of aggregation (i.e. differences in amino acid sequence, modifications of the protein resulting from aging, environmental factors) and to obtain information on aggregate structure. The third aim will test the hypothesis that the presence of chemical or molecular chaperones, or lipid vesicles that induce structure in a-synuclein in solution will inhibit the formation of insoluble fibrillar structures. The long-term goal of this aim is to provide a mechanism for the formation of Lewy bodies.