Z κ: AA transverse relaxation optimized spectroscopy NMR experiment measuring longitudinal relaxation interference

Abstract

NMR spin relaxation experiments provide a powerful tool for the measurement of global and local biomolecular rotational dynamics at subnanosecond time scales. Technical limitations restrict most spin relaxation studies to biomolecules weighing less than 10 kDa, considerably smaller than the average protein molecular weight of 30 kDa. In particular, experiments measuring z, the longitudinal HN1 - N15 dipole-dipole (DD) N15 chemical shift anisotropy (CSA) cross-correlated relaxation rate, are among those least suitable for use with larger biosystems. This is unfortunate because these experiments yield valuable insight into the variability of the N15 CSA tensor over the polypeptide backbone, and this knowledge is critical to the correct interpretation of most N15 -NMR backbone relaxation experiments, including R2 and R1. In order to remedy this situation, we present a new HN1 - N15 transverse relaxation optimized spectroscopy experiment measuring z suitable for applications with larger proteins (up to at least 30 kDa). The presented experiment also yields κ, the site-specific rate of longitudinal HN1 - H′1 DD cross relaxation. We describe the z κ experiment's performance in protonated human ubiquitin at 30.0 °C and in protonated calcium-saturated calmodulin/peptide complex at 20.0 °C, and demonstrate preliminary experimental results for a deuterated E. coli DnaK ATPase domain construct at 34 °C. © 2008 American Institute of Physics.