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

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

Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein

Misfolded a-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD). We present a high-resolution structure of an a-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique longrange distance restraints define a consensus structure with common amyloid features including parallel, in-register b-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of asynuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.This study was supported by the US National Institutes of Health (NIH) (grants R01-GM073770 to C.M.R., P50-NS053488 to V.M.Y.L. and P01-AG002132 to G.S.) and used SSNMR instrumentation procured with the support of grant S10-RR025037 (to C.M.R.) from the NIH National Center for Research Resources (NCRR). M.D.T., A.J.N. and A.M.B. were supported as members of the NIH Molecular Biophysics Training Grant at the University of Illinois at UrbanaChampaign (T32-GM008276), and D.J.C. is supported by grant T32-AG000255