TROSY IN TRIPLE-RESONANCE EXPERIMENTS - NEW PERSPECTIVES FOR SEQUENTIAL NMR ASSIGNMENT OF LARGE PROTEINS

The NMR assignment of C-13, N-15-labeled proteins with the use of trip le resonance experiments is limited to molecular weights below similar to 25,000 Daltons, mainly because of low sensitivity due to rapid tra nsverse nuclear spin relaxation during the evolution and recording per iods. For experiments that exclusively correlate the amide proton (H-1 (N)), the amide nitrogen (N-15), and C-13 atoms, this size limit has b een previously extended by additional labeling with deuterium (H-2). T he present paper shows that the implementation of transverse relaxatio n-optimized spectroscopy ([N-15,H-1]-TROSY) into triple resonance expe riments results in several-fold improved sensitivity for H-2/C-13/N-15 -labeled proteins and approximately twofold sensitivity gain for C-13/ N-15-labeled proteins, Pulse schemes and spectra recorded with deutera ted and protonated proteins are presented for the [N-15,H-1]-TROSY-HNC A and [N-15,H-1]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transve rse relaxation of N-15, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.