Measuring 13C/15N chemical shift anisotropy in [13C,15N] uniformly enriched proteins using CSA amplification

Abstract

Extended chemical shift anisotropy amplification (xCSA) is applied for measuring 13C/15N chemical shift anisotropy (CSA) of uniformly labeled proteins under magic-angle spinning (MAS). The amplification sequence consists of a sequence of π-pulses that repetitively interrupt MAS averaging of the CSA interaction. The timing of the pulses is designed to generate amplified spinning sideband manifolds which can be fitted to extract CSA parameters. The 13C/13C homonuclear dipolar interactions are not affected by the π-pulses due to the bilinear nature of the spin operators and are averaged by MAS in the xCSA experiment. These features make the constant evolution-time experiment suitable for measuring CSA of uniformly labeled samples. The incorporation of xCSA with multi-dimensional 13C/15N correlation is demonstrated with a GB1 protein sample as a model system for measuring 13C/15N CSA of all backbone 15NH, 13CA and 13CO sites.