The NMR assignment of 13 In the standard protocol for protein structure determination by NMR spectroscopy, sequence-specific resonance assignment plays a pivotal role (1). Several different assignment strategies are available, and one of the established procedures for obtaining sequential assignments (2) involves uniform 13 C͞ 15

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.

TROSY-type triple resonance experiments with the uniformly 2 H, 13 C, 15 N-labeled 7,8-dihydroneopterin aldolase (DHNA) from Staphylococcus aureus, which is a symmetric homooctamer protein of molecular mass 110 kDa, showed 20-fold to 50-fold sensitivity gains when compared to the corresponding conventional triple resonance NMR experiments. On this basis, sequential connectivities could be established for nearly all pairs of neighboring residues in DHNA. TROSY-type nuclear Overhauser enhancement spectroscopy yielded additional data to close the remaining gaps in the sequential assignment, and provided supplementary information on the secondary structure. Complete sequence-specific assignments of the 121-residue polypeptide chain in this 110 kDa octamer could thus be obtained in aqueous solution at 20°C, and the regular secondary structures in the solution conformation were found to coincide nearly identically with those in the crystal structure of the DHNA octamer.

The application of Ernst angle pulses in multidimensional NMR spectroscopy is theoretically and experimentally investigated. Theory shows that only for a few pulse sequences employed at high repetition rate, a remarkable gain in sensitivity is possible using Ernst angle pulses. As an example, a new variant of the heteronuclear multiple quantum coherence (HMQC) experiment, the fast-((1)H,(15)N)-HMQC, is described. This sequence allows, with a 1 mM protein sample in H(2)O, the acquisition of a highly resolved two-dimensional ((1)H,(15)N) correlated spectrum within 37 s. The high efficiency of the fast-HMQC to detect ligand binding to a target protein is demonstrated.

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A new program, MAPPER, for semiautomatic sequence-specific NMR assignment in proteins is introduced. The program uses an input of short fragments of sequentially neighboring residues, which have been assembled based on sequential NMR connectivities and for which either the 13C(alpha) and 13C(beta) chemical shifts or data on the amino acid type from other sources are known. MAPPER then performs an exhaustive search for self-consistent simultaneous mappings of all these fragments onto the protein sequence. Compared to using only the individual mappings of the spectroscopically connected fragments, the global mapping adds a powerful new constraint, which results in resolving many otherwise intractable ambiguities. In an initial application, virtually complete sequence-specific assignments were obtained for a 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13C alpha evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110 kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time period.

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