Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

Abstract

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 [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C 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 [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] 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 [superscript 15]N–[superscript 13]C 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 [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.