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The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13 C/ 15 N-resolved NOESY, CBCA(CO)NH, and 2D [ 13 C/ 15 N, 1 H]-HSQC were used, and 86.8% if exclusively 3D 13 C/ 15 N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa.

Polar side-chains in proteins play important roles in forming and maintaining three-dimensional structures, and thus participate in various biological functions. Until recently, most protein NMR studies have focused on the non-exchangeable protons of amino acid residues. The exchangeable protons attached to polar groups, such as hydroxyl (OH), sulfhydryl (SH), and amino (NH2) groups, have mostly been ignored, because in many cases these hydrogen atoms exchange too quickly with water protons, making NMR observations impractical. However, in certain environments, such as deep within the hydrophobic interior of a protein, or in a strong hydrogen bond to other polar groups or interacting ligands, the protons attached to polar groups may exhibit slow hydrogen exchange rates and thus become NMR accessible. To explore the structural and biological implications of the interactions involving polar side-chains, we have developed versatile NMR methods to detect such cases by observing the line shapes of (13)C NMR signals near the polar groups, which are affected by deuterium-proton isotope shifts in a mixture of H2O and D2O. These methods allow the detection of polar side-chains with slow hydrogen-deuterium exchange rates, and therefore provide opportunities to retrieve information about the polar side-chains, which might otherwise be overlooked by conventional NMR experiments. Future prospects of applications using deuterium-proton isotope shifts to retrieve missing structural and dynamic information of proteins are discussed.

We describe the construction and performance of an NMR tube with a magnetic susceptibility matched sample cavity that confines the solution within the detection zone in the axial direction and in a quasi-rectangular region in the radial direction. The slot-like sample cavity provides both good sample volume efficiency and tolerance to sensitivity loss in the sample space. The signal-to-noise ratio per unit volume of the constructed tube was 2.2 times higher than that of a cylindrical tube of 5 mm outer diameter with a sample containing 300 mM NaCl at a static magnetic field of 14.1 T. Even the overall signal-to-noise ratio of the slot tube was 35% higher than that of the conventional 5 mm tube for a sample containing 300 mM NaCl. Similar improvements over existing sample tube geometries were obtained at 950 MHz. Moreover the temperature rise resulting from RF heating was found to be significantly lower for the slot tube even when compared to 3 and 4 mm outer diameter cylindrical tubes as measured in a 5 mm cryoprobe. A further advantage of this type of tube is that a sample cavity of any desired size and shape can be formed within a cylindrical tube for use in a single cryogenic probe.

In order to determine precise three-dimensional structures of proteins by residual dipolar coupling constants as the major or even exclusive structural constraints, it is essential to use two anisotropic media. In doing so, a reliable and versatile method for estimating the relative orientation of the alignment tensors for the molecules dissolved in different anisotropic media is required. In this communication, we present a new graphical approach for this purpose, which does not require structural information of the target molecules. The correlation map for the two independent data sets of residual dipolar coupling constants, which can be obtained for the molecules in different anisotropic media, strongly depends on the relative orientation of the alignment tensors. We have simulated the correlation maps for all possible combinations of the Euler angles, which transform one alignment tensor to the other, and compared them to the experimental data sets reported for labeled human ubiquitin. This simple graphical method affords a useful starting point for the structural determinations using residual dipolar couplings.

In 1 H– 15 N TROSY experiments of proteins and nucleic acids, where the second coherence transfer delay time τ′ has been fixed as 5.6 ms, 1/(2 1 J NH ), in order to achieve complete spin-state selection, spurious negative peaks are observed along the 15 N axes. These peaks are often annoyingly large, especially for nucleic acids. A simple product operator calculation, however, indicated that the shortening of the second delay time τ′, which is next to the t 1 period, would efficiently suppress these spurious peaks, without sacrificing the sensitivities of the TROSY peaks too much. We have shown for three systems, two 11- and 17-kDa proteins and one 8-kDa DNA duplex, that these spurious peaks can be effectively suppressed with delay times of 3.3 ms for the two proteins and 2.3 ms for the DNA. These delay times, optimized by trial and error, for the spurious peak suppression did not depend on the magnetic field strength and the temperature very much. Although the shortened τ′ delay times attenuate the TROSY peak intensities by about 10 and 20% for the two proteins and the DNA, respectively, this simple modification will be useful for the quantitative uses of TROSY peaks and will result in cleaner spectra for various TROSY-based multiple resonance experiments.

A two-dimensional [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment (2D [31P]-sedct-[13C, 1H]-HMQC) is introduced for measurements of 3J(C4'P) and 3J(H3'P) scalar couplings in large 13C-labeled nucleic acids and in DNA-protein complexes. This experiment makes use of the fact that 1H-13C multiple-quantum coherences in macromolecules relax more slowly than the corresponding 13C single-quantum coherences. 3J(C4'P) and 3J(H3'P) are related via Karplus-type functions with the phosphodiester torsion angles beta and epsilon, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly 13C, 15N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein-DNA complex.