This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. PrPC is a membrane-associated protein believed to be involved in cellular adhesion and signaling. Furthermore, the protein also exists in an infectious form that is linked to diseases such as Bovine Spongiform Encephalopathy ("mad cow disease") and Creutzfeldt?Jakob disease. This infectious isoform, termed PrPSc, is also known as a prion. PrPSc is capable of acting as an infectious agent through templating of the misfolded state onto PrPC. The precise conversion mechanism how PrPC is converted to PrPSc, and how subsequently amyloid fibrils are formed, are currently unknown. Several potential intermediates have been identified, including a mainly ?-sheeted oligomer and several polymers. Using a recombinantly expressed hamster PrP as a model system, it has been shown that some of these multimers can be separated by size exclusion chromatography (SEC). However, due to the tendency of PrP to rapidly aggregate, these different forms have yet to be characterized structurally. We propose to characterize these isoforms using solution x-ray scattering at bio-SAXS beamline BL4-2. We have developed a new protocol for connecting a HPLC size exclusion column (SEC) column directly to the BL4-2 scattering instrument, allowing rapid analysis of the protein fractions as they are eluted. We have already succeeded in characterizing the normal physiological form of recombinant PrP using this HPLC setup (preliminary results discussed below), and we now propose to refine our protocol to analyze the other isoforms and study initial oligomeric assembly intermediates leading to the formation of infectious amyloid fibers. Additionally we will also explore the effects of varying the protein buffer and the addition of small compounds on PrPC using the BL4.2 high-throughput facilities.