Tuesday, August 16, 2011

Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

Bayro, M. J., Debelouchina, G. T., Eddy, M. T., Birkett, N. R., MacPhee, C. E., Rosay, M., Maas, W. E., Dobson, C. M., and Griffin, R. G. (2011) Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR, J. Am. Chem. Soc.


We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range 13C-13C correlation spectra and the third based on the identification of intermolecular interactions in 13C-15N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient 13C-13C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The 13C-13C experiments are corroborated by 15N-13C correlation spectra obtained from a mixed [15N,12C]/[14N,13C] 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 15N-13C 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 problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular 13C-13C and 15N-13C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.