TROSY-type Triple-Resonance Experiments for Sequential NMR Assignments of Large Proteins

Abstract

Transverse relaxation-optimized spectroscopy (TROSY) was implemented in the four triple resonance experiments [15N,1H]-TROSY-HN(CO)CA, [15N,1H]-TROSY-HN(CA)CO, [15N,1H]-TROSY-HNCACB, and [15N,1H]-TROSY-HN(CO)CACB. Combined with [15N,1H]-TROSY-HNCA and [15N,1H]-TROSY-HNCO (Salzmann, M.; Pervushin, K.; Wider, G.; Senn, H. Wüthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1998, 13585−13590) these experiments represent a suite of TROSY-type triple resonance experiments that enables sequential backbone assignment of proteins. When used with the 23 kDa 2H/13C/15N-labeled protein gyrase 23B, a comparison with the corresponding conventional NMR experiments showed, on average over the entire amino acid sequence, a 3-fold sensitivity gain for each of the four experiments. The use of the TROSY principle in triple resonance experiments thus promises to enable resonance assignments for significantly larger proteins than what is achievable today with the corresponding conventional NMR experiments.