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 [ 15 N, 1 H]-TROSY-HN(CO)CA, [ 15 N, 1 H]-TROSY-HN(CA)CO, [ 15 N, 1 H]-TROSY-HNCACB, and [ 15 N, 1 H]-TROSY-HN(CO)CACB. Combined with [ 15 N, 1 H]-TROSY-HNCA and [ 15 N, 1 H]-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 2 H/ 13 C/ 15 N-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.

Structure-Function Analysis of a Broad Specificity Populus trichocarpa Endo-β-glucanase Reveals an Evolutionary Link between Bacterial Licheninases and Plant XTH Gene Products

“…Matrix (36,37), HNCO (38), and HN(CA)CO (37) spectra were processed with NMRPipe (39) and analyzed with Sparky (40). The 1 H and 15 N dimensions were corrected by ϩ46 Hz and Ϫ46 Hz, respectively, to adjust for the TROSY shift.…”

Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

“…Sequential assignments of H N , N, CЈ, C ␣ , and C ␤ resonances of apo-and holo-HisJ, D1, and D2 were achieved using two-dimensional 1 H, 15 N-HSQC and three-dimensional HNCACB, HN(CO)CACB, HN(CA)CO, and HNCO experiments. For apo-and holo-HisJ and D1 proteins, TROSY versions of these experiments were used (29,30). For apo-HisJ, methyl group assignments were acquired using a three-dimensional C-TOCSY-CHD 2 experiment (28 15 N frequency (32,33).…”

Conformational stability and activity of p73 require a second helix in the tetramerization domain

“…32 Protein backbone resonances were assigned with TROSY-based HNCACB, HNCOCA and HNCO experiments. 33,34 Side-chain resonances were assigned with H(CCCO)NH, (H)C(CCO)NH, 3D TOCSY-[ 15 N, 1 H]-TROSY and 13 C-separated 3D NOESY experiments. The prochiral methyl groups of valine and leucine with a CT-[ 13 C, 1 H]-HSQC were assigned by 10% nonrandom 13 C protein labeling.…”