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

The 2 H, 13 C, 15 N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13 C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX͞DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein.The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.A bout one-third of the genes in living organisms are assumed to encode integral membrane proteins (e.g., refs. 1-3), and three-dimensional (3D) structure determination of this class of proteins is fundamental to the understanding of a wide spectrum of biological functions. Notwithstanding the crucial importance of work in this area, the database of 3D membrane protein structures is still small, which reflects the challenge presented by this class of molecules to structural biologists. In particular, the solution NMR techniques that commonly are applied with biological macromolecules (e.g., refs. 4 and 5) so far only in few instances have been used with membrane proteins (e.g., refs. 6 and 7) or membrane-binding polypeptides (e.g., refs. 8 and 9), whereby appropriate detergents were used to keep the proteins in solution. Suitable micelles for such studies must ensure the structural and functional integrity of the membrane protein, should be a good mimic of the natural environment in the cell membrane, and need to be sufficiently small to allow rapid Brownian motions of the mixed micelles in solution (e.g., refs. 10-12). The large size of the structures obtained upon reconstitution and solubilization of membrane proteins in detergent micelles actually has limited the application of solution NMR techniques to such systems because of the slow tumbling in solution and the concomitantly large linewidths. New NMR techniques are now available to extend the size limits for NMR in solution, i.e., transverse relaxation-optimized spectroscopy (TROSY) (13) and cross-correlated relaxation-enhanced polarization transfer (CRINEPT) (14), and high-quality NMR spectra have been presented for protei...

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 39 ends. The La protein binds the 39 ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4D cells requires Rex1p, these pretRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4D cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4D cells. Our experiments reveal that one consequence of Rex1p-dependent 39 trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.

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, which is centered about the middle of the long axis through the ␤-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the ␤-barrel in a doublelayer lipid membrane.NOE ͉ detergents ͉ solvation of membrane proteins ͉ intermolecular NOEs M embrane proteins constitute about one-third of all proteins encoded by the genomes of living organisms. However, they are strongly underrepresented in the database of 3D protein structures, which reflects the big challenge presented by this class of proteins to structural biologists. Apart from difficulties related to high-yield expression, purification, and refolding, considerable additional effort is usually required for finding either suitable crystallization conditions or solution conditions for NMR measurements, whereby detergent micelles, bicelles, lipid bilayers, or lipid vesicles are commonly used as a replacement of the natural membrane environment (1-4). For solution NMR studies, the overall size of the mixed protein͞deter-gent͞lipid supramolecular structure is an important factor, in addition to the preservation of the natural structure and function of the protein, and this combined demand has most promisingly been met with protein-detergent micelles. The use of transverse relaxation-optimized spectroscopy (TROSY) (5-9) and advanced isotope labeling strategies (10) has now actually opened avenues for NMR structure determination of integral membrane proteins reconstituted in detergent micelles (1,(11)(12)(13). In this context, the nature of the protein-detergent interactions in mixed micelles is of keen interest.Various different schemes for the interaction between detergent molecules and membrane proteins have been suggested (14,15). Based on lipid binding quantification by chromatographic methods and model calculations on sarcoplasmic reticulum Ca 2ϩ -ATPase, Møller and le Maire (14) proposed that formation of a monolayer rather than a bilayer type of interaction is the basis for solubilization of membrane proteins by detergents. With reference to unfolding studies of the Escherichia coli outer membrane protein OmpA in micelles formed by detergent molecules with different chain lengths, Kleinschmidt et al. (15) advanced the idea that a monolayer or a prolate ellipsoid arrangement of detergent molecules on the hydrophobic protein surface prevails in the mixed micelles. In this paper, we use solution NMR spectroscopy for further experimental studies of membrane protein-detergent interactions.The potentialities of high-resolution NMR spectroscopy for studies of the architecture of mixed polypeptide-detergent micelles have long been recognized (16)(17)(18)(19), and the technique also has recently been applied with membrane protein fragments (20-22).Here, we present a study of the intact integral...

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For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13 C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA L L-barrel from those of OmpX. Overall, the present data demonstrate that relaxationoptimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins. ß