Projects per year
Abstract / Description of output
We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this urpose, two being based on the analysis of long-range C-13-C-13 correlation spectra and the third based on the identification of intermolecular interactions in C-13-N-15 spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (P13-SH3 or PI3K-SH3), that efficient C-13-C-13 correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the Eroteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The C-13-C-13 experiments are corroborated by N-15-C-13 correlation spectra obtained from a mixed [N-15,C-12]/[N-14,(13)k.,] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular N-15-C-13 constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a roblem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular C-13-C-13 and N-15-C-13 constraints that establish that the P13-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.
Keywords / Materials (for Non-textual outputs)
- SOLID-STATE NMR
- BETA-SHEET STRUCTURE
- ROTATING SOLIDS
- PRION PROTEIN
FingerprintDive into the research topics of 'Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR'. Together they form a unique fingerprint.
- 1 Finished
1/09/07 → 31/05/10