Attenuated T2 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

Fast transverse relaxation of ¹H, ¹⁵N, and ¹³C 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 ¹H,¹⁵N-correlation experiment with a uniformly ¹⁵N-labeled protein in a DNA complex of molecular mass 17 kDa at a ¹H frequency of 750 MHz showed that ¹⁵N relaxation during ¹⁵N chemical shift evolution and ¹H^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 ¹H,¹⁵N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for ¹⁵N and 15 Hz for ¹H^N at 4 degrees 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 ¹⁵N-labeled protein of 150 kDa at 750 MHz and 20 degrees C one predicts residual linewidths of 10 Hz for ¹⁵N and 45 Hz for ¹H^N, and for the corresponding uniformly ¹⁵N,²H-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.