Attenuated T 2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Abstract

Fast transverse relaxation of 1 H, 15 N, and 13 C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1 H, 15 N-correlation experiment with a uniformly 15 N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1 H frequency of 750 MHz showed that 15 N relaxation during 15 N chemical shift evolution and 1 H N relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1 H, 15 N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15 N and 15 Hz for 1 H N at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15 N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15 N and 45 Hz for 1 H N , and for the corresponding uniformly 15 N, 2 H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.