Measurement of (3h)J(NC ') connectivities across hydrogen bonds in a 30 kDa protein

A method is described which permits detection of (3h)J(NC') scalar coupling s across hydrogen bonds in larger, perdeuterated proteins. The experiment i s demonstrated for the uniformly H-2/C-13/N-15-enriched 30 kDa ribosome ina ctivating protein MAP30. The (3)hJ(NC') interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the N-15 chemical shift anisotropy an d H-1-N-15 dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depe nds strongly on the N-15 transverse relaxation rate of the downfield N-15 m ultiplet component and on the amide proton T-1. In perdeuterated MAP30 at 4 0 degrees C, the average TROSY T-2 was 169 ms at 750 MHz H-1 frequency, and a wide range of longitudinal relaxation rates was observed for the amide p rotons.