Solid-State Nuclear Magnetic Resonance Spectroscopy of Alpha-Synuclein Fibrils

Abstract

181 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.Recent solid-state NMR (SSNMR) studies on protein systems such as beta-amyloid and the prion HET-s demonstrate the applicability of these techniques towards solving 3D structures of amyloid fibrils. Alpha-synuclein is a 140-residue protein that has been implicated in Parkinson's disease. Fibrils of alpha-synuclein are the primary fibrillar component of Lewy bodies, which are the pathological hallmark of the disease; however, the role that synuclein plays in the disease is uncertain, due largely to a lack of atomic-resolution structural information. Here we present structural studies of fibrils of alpha-synuclein. In this work, we first optimized an expression and purification scheme for the production of uniformly 13C and 15N labeled synuclein. We then developed a protocol for the preparation of reproducible fibril samples by the use of pre-formed fibril seeds. Two of the mutant forms of synuclein that have been implicated in early-onset forms of Parkinson's disease were evaluated for dynamic and structural differences relative to fibrils of wild-type. For fully hydrated fibrils, optimal sensitivity was achieved at lower temperatures, likely due to decreased motion in the mobile regions. Nearly complete 13C and 15N chemical shift assignments were made for the core region of the fibril. Spectral sensitivity was increased by drying the fibril samples prior to packing. SSNMR chemical shift analysis was used to assess perturbations in the dried fibril samples; differences were minimal, indicating that bulk water is not necessary to maintain the structure of the fibril core. We then further enhanced spectral resolution by developing a method for partially hydrating fibril samples. These rehydrated samples dramatically improve spectral resolution with a minimal sacrifice in overall signal intensity; thus, they offer the best compromise between resolution and sensitivity. We have also incorporated selective labeling schemes to facilitate determination of distance restraints, a requirement for a three-dimensional, atomic-resolution structure. We also report on progress in the interpretation of multidimensional SSNMR spectra of these samples as well as initial medium and long-range distances determined from these spectra. Overall, the work presented in this dissertation demonstrates the potential for solving high-resolution structures of synuclein fibrils using solid-state NMR techniques.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD