Marc Baldus

The 140-residue protein alpha-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly (13)C/(15)N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. (13)C and (15)N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the (15)N dimension and up to 5 ppm for backbone and side-chain (13)C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to beta-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution.

A frequency selective heteronuclear polarization transfer technique is introduced for rotating solids. In this method, radiofrequency fields comparable with the frequency offsets are applied to establish Hartmann-Hahn cross polarization that therefore depends explicitly on the resonance offset of the nuclei involved. Under these conditions, spectrally induced filtering in combination with cross polarization (SPECIFIC CP) can be achieved and is demonstrated to be useful for spectral simplification or assignment in heteronuclear spin pairs. The design principles are outlined and demonstrated experimentally on 13C, 15N labelled amino acids.

The backbone and side-chain 13C and 15N signals of a solid 62-residue (u-13C,15N)-labelled protein containing the alpha-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) 15N-13C and 13C-13C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D 13C-13C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton-nitrogen and a nitrogen-carbon cross-polarisation step followed by a carbon-carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7-61, while the signals of the N- and C-terminal residues 1-6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.