NMR spectroscopy with proteins based on observation of a small number of spins with outstanding spectral properties, which either may be present naturally or introduced by techniques such as site-specific isotope labeling, yielded biologically relevant information on human hemoglobin (M ϭ 65,000) as early as 1969 (1), and subsequently also for significantly larger systems such as Igs (2). In contrast, the use of NMR for de novo structure determination (3, 4) so far has been limited to relatively small molecular sizes, with the largest NMR structure below molecular weight 30,000. Although NMR in structural biology may, for practical reasons of coordinated use with x-ray crystallography (5), focus on smaller molecular sizes also in the future, considerable effort goes into attempts to extend the size limit to bigger molecules (for example, see refs. 6-8). Here we introduce transverse relaxation-optimized spectroscopy (TROSY) and present experimental data and theoretical considerations showing that this approach is capable of significantly reducing transverse relaxation rates and thus overcomes a key obstacle opposing solution NMR of larger molecules (7).At the high magnetic fields typically used for studies of proteins and nucleic acids, chemical shift anisotropy interaction (CSA) of 1 H, 15 N, and 13 C nuclei forms a significant source of relaxation in proteins and nucleic acids, in addition to dipole-dipole (DD) relaxation. This leads to increase of the overall transverse relaxation rates with increasing polarizing magnetic field, B 0 . Nonetheless, transverse relaxation of amide protons in larger proteins at high fields has been reduced successfully by complete or partial replacement of the nonlabile hydrogen atoms with deuterons and, for example, more than 90% of the 15 N, 13 C ␣ , and 1 H N chemical shifts thus were assigned in the polypeptide chains of a protein-DNA complex of size 64,000 (6). TROSY uses spectroscopic means to further reduce T 2 relaxation based on the fact that cross-correlated relaxation caused by DD and CSA interference gives rise to different relaxation rates of the individual multiplet components in a system of two coupled spins 1 ⁄2, I and S, such as the 15 N-1 H fragment of a peptide bond (9, 10). Theory shows that at 1 H frequencies near 1 GHz nearly complete cancellation of all transverse relaxation effects within a 15 N-1 H moiety can be achieved for one of the four multiplet components. TROSY observes exclusively this narrow component, for which the residual linewidth is then almost entirely because of DD interactions with remote hydrogen atoms in the protein. These can be efficiently suppressed by 2 H-labeling, so that in TROSY-type experiments the accessible molecular size for solution NMR studies no longer is primarily limited by T 2 relaxation. TheoryWe consider a system of two scalar coupled spins 1 ⁄2, I and S, with a scalar coupling constant J IS , which is located in a protein molecule. T 2 relaxation of this spin system is dominated by the DD coupling of I and S a...

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

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

H]-transverse relaxation-optimized spectroscopy (TROSY) (3-5) of scalar couplings across the Watson-Crick base pairs in isotope-labeled DNA, which affords direct observation of the hydrogen bonds in these structures. Scalar couplings across hydrogen bonds have been previously reported for organicsynthetic compounds (6, 7), RNA fragments (8), and a metalloprotein (9, 10). The variability of such couplings observed so far indicates that they may become sensitive new parameters for detection of hydrogen bond formation and associated subtle conformational changes. Furthermore, in conjunction with quantum-chemical calculations, precise measurements of scalar couplings across hydrogen bonds can be expected to provide novel insights into the nature of hydrogen bonds in chemicals and in biological macromolecules. MATERIALS AND METHODSFully and partially 13 C, 15 N-doubly labeled DNA oligomers were synthesized on a DNA synthesizer (Applied Biosystems model 392-28) by the solid-phase phosphoroamidite method, by using isotope-labeled monomer units that had been synthesized according to a previously described strategy (11). Approximately 1 mol of oligomer was obtained from 5 mol of nucleoside bound to the resin. NMR samples of the DNA duplex at a concentration of Ϸ2 mM were prepared in 90% H 2 O͞10% D 2 O containing 50 mM potassium phosphate and 20 mM KCl at pH 6.0. NMR measurements were performed at 15°C on Bruker DRX500 and DRX750 spectrometers equipped with H bond length, the solid-state NMR value of 0.11 nm for G and T in a hydrated DNA duplex (19) was used. Relaxation of the imino proton due to dipole-dipole (DD) coupling with remote protons in the DNA duplex was represented as follows (2): in the Watson-Crick AAT pair by an adenosine amino proton at a distance of 0.24 nm and the adenosine C2 proton at 0.3 nm; in G'C by a guanosine amino proton at 0.22 nm and a cytosine amino proton at 0.25 nm. For both base pairs, two imino protons in sequentially stacked bases at 0.4 nm also were considered. Following the calculations outlined in refs. 3-5, the use of TROSY at a polarizing magnetic field B o ϭ 17.6 T is expected to yield 65% and 30% reductions of the 15 N and 1 H linewidth, respectively, for AAT base pairs and 55% and 20% reductions for G'C base pairs. If the contributions from dipolar interactions with remote protons are neglected, the calculations predict reductions of 85% and 75% for 15 N and 1

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a [Formula: see text] sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the 'water flip back' technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.

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.

Transverse relaxation-optimized spectroscopy (TROSY) yields greatly improved sensitivity for multidimensional NMR experiments with aromatic spin systems in proteins. TROSY makes use of the fact that due to the large anisotropy of the 13C chemical shift tensor, the transverse relaxation of one component of the 13C doublet in aromatic 13C−1H moieties is reduced by interference of dipole−dipole (DD) coupling and chemical shift anisotropy (CSA) relaxation. The full advantage of TROSY for studies of aromatic spin systems is obtained at presently available resonance frequencies from 500 to 800 MHz. Since the 13C chemical shifts are recorded using a constant-time evolution period, the TROSY improvement in signal-to-noise relative to corresponding conventional NMR experiments increases with increasing molecular size and can be further significantly enhanced by combined use of the 1H and 13C steady-state magnetizations.With selective observation of the slowly relaxing component of the 13C doublets in experiments recorded without 1H decoupling during the 13C chemical shift evolution period, a 4−10-fold sensitivity gain for individual aromatic 13C−1H correlation peaks was achieved for the uniformly 13C-labeled 18 kDa protein cyclophilin A. A new 3D ct-TROSY-HCCH-COSY experiment is presented, which correlates the resonances of 13C nuclei with those of covalently bound 13C−1H groups and can be applied for complete identification of aromatic spin systems. In this scheme the chemical shift evolution of neighboring aromatic 13C spins are recorded in two indirectly detected spectral dimensions, so that the additional third dimension is obtained without increase of the number of delays.

In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings [insensitive nuclei enhanced by polarization transfer (INEPT)] are key elements of the pulse schemes. For molecular weights beyond 100,000, transverse relaxation during the transfer time may become a limiting factor. This paper presents a transfer technique for work with big molecules, cross relaxationenhanced polarization transfer (CRINEPT), which largely reduces the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. Structure determination of proteins by NMR in solution (1) has so far been limited to the molecular weight range up to Ϸ30 kDa (2), and experiments yielding backbone assignments for 2 H-labeled proteins up to Ϸ60 kDa have been reported (3). In larger molecular species, the standard experimental techniques (4, 5) lead to severe sensitivity loss because of transverse relaxation even when optimal isotope-labeling is used (6). The situation has recently been improved with the introduction of the transverse relaxation-optimized spectroscopy (TROSY) technique, which reduces transverse relaxation during evolution and observation periods in heteronuclear NMR experiments (7,8). For example, for 15 NO 1 H-moieties in proteins, significant reduction of transverse relaxation during the 15 N evolution and amide proton acquisition periods can be achieved at the highest presently available 1 H frequencies, and nearly complete cancellation is expected at 1 H frequencies near 1 GHz (7, 9). TROSY already has been applied for detailed NMR studies of a protein with molecular weight Ͼ100 kDa (8). Theoretical considerations indicate that TROSY will reach its limits at somewhat larger sizes because of rapid transverse relaxation during the insensitive nuclei enhanced by polarization transfer (INEPT)-type (10) magnetization transfers via scalar couplings between the different nuclei. This paper presents a transfer technique for work with very large molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which is based on combining cross-correlated relaxation-induced polarization transfer (11-14) and polarization transfer via scalar couplings (10). To provide a foundation for practical applications of CRINEPT, the performance of the previously little-used cross relaxation-induced polarization transfer (CRIPT) is investigated for 15