The long-term goal of this proposal is development of a detailed kinetic description of the initiation of amyloid aggregation, a common feature of the diverse group of devastating diseases characterized by the deposition of fibrillar aggregates of misfolded proteins. The focus is a description of the identities and functions of the nascent intermediate molecular species as a means for identifying target protein species for the development of methods of controlling the aggregation pathway. The specific proposed hypothesis is that non-fibrillar oligomeric protein species in conjunction with conformational changes in the native proteins play the crucial role in the initiation of aggregation of amyloid-forming proteins. This hypothesis is based on evidence that 1) soluble oligomers of amyloid proteins are known to be cytotoxic and 2) solutions influencing protein secondary structure of amyloid proteins affect their aggregation kinetics. The experimental strategies rely on modern biophysical instrumentations based on multiphoton microscopy and fluorescence correlation spectroscopies to characterize the solution and lipid-associated behavior of three key amyloid-forming proteins: alpha-Synuclein, A-beta, and apomyoglobin.
Two specific aims are: 1. Quantification and understanding of the conformational changes associated with the initiation of aggregation utilizing adaptations of fluorescence correlation spectroscopy to characterize protein conformational fluctuations and folding/misfolding associated conformational changes in apomyoglobin, alpha-Synuclein, and A-beta. 2. Biophysical characterization of the soluble oligomers formed by alpha- Synuclein and A-beta, including (i) their solution equilibrium properties, and (ii) the nature of the interactions with lipid membranes, in particular the role of membrane charge and lipid-ordered phase formation, to understand how cell membrane interactions affect aggregation. ? ?
