TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

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