Protein Structure Characterization by Solid-State NMR: Structural Comparison of Mouse and Human alpha-Synuclein Fibrils, Sparse 13C Labeling Schemes, and Stereospecific Assignment of Val and Leu Prochiral Methyl Groups

Abstract

This thesis describes protein structure characterization by solid-state nuclear magnetic resonance (ssNMR), including structural investigation of mouse alpha-synuclein (maS) fibrils and the comparison to human apha-synuclein (aS) fibrils, sparse 13C labeling schemes based on [1-13C]Glucose ([1-13C]Glc) and [2-13C]Glucose ([2-13C]Glc), and stereospecific assignment of Val and Leu prochiral methyl groups by the [2-13C]Glc labeling scheme. Fibrillar aS is the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease. maS aggregates much faster than haS, although maS differs from haS at only seven positions in its primary sequence. Currently, little is known about the site-specific structural differences between maS and haS fibrils. In Part I of this thesis, we applied state-of-the-art ssNMR methods to structurally characterize maS fibrils. The assignment strategy employed a set of high-resolution 2D and 3D ssNMR spectra recorded on uniformly [13C]Glc-, [1-13C]Glc-, and [2-13C]Glc-labeled maS fibrils. Proteins produced with [1-13C]Glc or [2-13C]Glc are very sparsely labeled and the resulting 2D ssNMR spectra exhibit smaller linewidths and contain a reduced number of cross peaks. This allows for an accelerated and straightforward resonance assignment using a limited set of simple 2D experiments. The approach is successfully demonstrated with the de novo assignment of maS fibrils. The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. In Part II of this thesis, we present a straightforward method for the stereospecific ssNMR assignment of the prochiral methyl groups of Val and Leu using [2-13C]Glc as the sole carbon source for the biosynthesis of amino acids. The approach is fundamentally based on the stereoselective biosynthetic pathway of Val and Leu, and the co-presence of [2-13C]pyruvate produced mainly by glycolysis and [3-13C]/[1,3-13C]pyruvate most probably formed through scrambling in the pentose phosphate pathway starting from [2-13C]Glc. As a consequence, the isotope spin pairs of 13Cb-13Cg2 and 13Ca-13Cg1 in Val, and 13Cg-13Cd2 and 13Cb-13Cd1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and of microcrystalline ubiquitin.