Project Summary: The goal of this proposal is to characterize rare conformations of the Parkinson's disease- linked protein alpha-synuclein (aS). aS is a small (140 kD) protein that has been found to comprise the main component of Lewy bodies, the primary pathological indicator of Parkinson's disease. Within Lewy Bodies, aS aggregates in the form of amyloid fibrils, which can also be formed from the protein in vitro via a nucleation dependent polymerization process. The free state of aS samples a predominantly disordered conformational ensemble in solution, but the protein adopts a highly ordered (3-sheet rich structure in the fibril state. Recent evidence suggests that fast conformational exchange processes in the free state of the protein may control entry into the fibril formation pathway. Our preliminary data indicate that this exchange occurs predominantly between a major highly unfolded conformational ensemble and a minor conformation that may be more structured. Here we propose to use recently developed high-resolution solution state NMR relaxation dispersion measurements to characterize this minor conformation. We will measure the rates, populations, and chemical shift changes associated with conformational exchange between the two conformations at the level of individual residues. The results will elucidate the detailed structural changes that accompany the earliest steps in the conversion of the protein from its soluble monomeric form to a putative precursor for subsequent toxic oligomeric and fibrillar species. The effects of specific point mutations associated with early onset Parkinson's disease on the conformational exchange process and on the structural properties of the rare conformation will also be studied in the context of the known effects of these mutations on fibril formation. Relevance to Health: The mechanism of amyloid fibril formation is poorly understood, especially on a structural level, but it appears that intermediates on this pathway play an important part the development of Parkinson's disease. We expect that a structural description of intermediate forms of aS that likely precede and are necessary for the fibrillization process will facilitate the development of improved therapeutic strategies for treating the disease. Furthermore, amyloid fibril formation is a feature of several other neurodegenerative diseases including Alzheimer's, Huntington's and possibly ALS. Therefore a characterization of the fibrillization pathway of Parkinson's disease may well provide general insights into the amyloid formation pathways of these other important disorders.