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

East transverse relaxation of H-1, N-15, and C-13 by dipole-dipole cou pling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacr omolecular structures that can be studied by NMR spectroscopy in solut ion, Transverse relaxation-optimized spectroscopy (TROSY) is an approa ch for suppression of transverse relaxation in multidimensional NMR ex periments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional H-1, N- 15-correlation experiment with a uniformly N-15-labeled protein in a D NA complex of molecular mass 17 kDa at a H-1 frequency of 750 MHz show ed that N-15 relaxation during N-15 chemical shift evolution and H-1(N ) relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions, The reduction o f the linewidths when compared with a conventional two-dimensional H-1 , N-15-correlation experiment was 60% and 40%, respectively, and the r esidual linewidths were 5 Hz for N-15 and 15 Hz for H-1(N) at 4 degree s C, Because the ratio of the DD and CSA relaxation rates is nearly in dependent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger protei ns, For a N-15-labeled protein of 150 kDa at 750 MHz and 20 degrees C one predicts residual linewidths of 10 Hz for N-15 and 45 Hz for H-1(N ), and for the corresponding uniformly N-15, H-2-labeled protein the r esidual linewidths are predicted to be smaller than 5 Hz and 15 Hz, re spectively, The TROSY principle should benefit a variety of multidimen sional solution NMR experiments, especially with future use of yet som ewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.