Simultaneous measurement of 1H�C15N and Methyl 1Hm�C13Cm residual dipolar couplings in large proteins

A two-dimensional TROSY-based SIM-(13)C(m)-(1)H(m)/(1)H-(15)N NMR experiment for simultaneous measurements of methyl (1) D (CH) and backbone amide (1) D (NH) residual dipolar couplings (RDC) in {U-[(15)N,(2)H]; Ileδ1-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-(13)C(m)-(1)H(m)/(1)H-(15)N data sets provide convenient means of establishing the alignment tensor characteristics via the measurement of (1) D (NH) RDCs in the same protein sample.     

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.     

A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on ¹⁵N, natural abundance ¹³C samples. The experiment exploits the fact that transfers of magnetization from ¹⁵N to ¹³CO and from ¹⁵N to ¹³C (and back) proceed independently for samples that are not enriched in ¹³C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM ¹⁵N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.     

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.     

A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

Summary A general approach for assigning the resonances of uniformly ¹⁵N- and ¹³C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The ¹H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D ¹⁵N-edited ¹H, ¹H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete ¹³C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the ¹H, ¹³C, and ¹⁵N resonances of the FK506 binding protein denatured in 6.3 M urea.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure–function studies

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure–function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. ¹H and ¹³C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment (¹H^N, ¹⁵N, ¹³CO, ¹Hα/¹³Cα, and ¹Hβ/¹³Cβ chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca²⁺ bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.     

Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR

We have expressed [U-(13)C,(15)N]-labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11 mg/l of growth medium. Nuclear magnetic resonance (NMR) studies were conducted on the Fe(3+) form of the protein. We report herein chemical shift assignments for amide (1)H and (15)N, (13)C(omicron), (13)C(alpha), (13)C(beta), (1)H(alpha) and (1)H(beta) resonances based upon a series of three-dimensional NMR experiments: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HCA(CO)N, HCCH-TOCSY and HBHA(CBCA)NH. An investigation of the chemical shifts of the threonine residues was also made by using density functional theory in order to help solve discrepancies between (15)N chemical shift assignments reported in this study and those reported previously.     

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.     

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

The prediction of protein structural class using averaged chemical shifts

Knowledge of protein structural class can provide important information about its folding patterns. Many approaches have been developed for the prediction of protein structural classes. However, the information used by these approaches is primarily based on amino acid sequences. In this study, a novel method is presented to predict protein structural classes by use of chemical shift (CS) information derived from nuclear magnetic resonance spectra. Firstly, 399 non-homologue (about 15% identity) proteins were constructed to investigate the distribution of averaged CS values of six nuclei ((13)CO, (13)Cα, (13)Cβ, (1)HN, (1)Hα and (15)N) in three protein structural classes. Subsequently, support vector machine was proposed to predict three protein structural classes by using averaged CS information of six nuclei. Overall accuracy of jackknife cross-validation achieves 87.0%. Finally, the feature selection technique is applied to exclude redundant information and find out an optimized feature set. Results show that the overall accuracy increased to 88.0% by using the averaged CSs of (13)CO, (1)Hα and (15)N. The proposed approach outperformed other state-of-the-art methods in terms of predictive accuracy in particular for low-similarity protein data. We expect that our proposed approach will be an excellent alternative to traditional methods for protein structural class prediction.     

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and C alpha H protons in this largely alpha-helical protein. We demonstrate that the combined use of four new types of heteronuclear 3D NMR spectra together with the previously described HOHAHA-HMQC 3D experiment [Marion, D., et al. (1989) Biochemistry 28, 6150-6156] can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D NMR experiments use 1H detection and rely on multiple-step magnetization transfers via well-resolved one-bond J couplings, offering high sensitivity and requiring a total of only 9 days for the recording of all five 3D spectra. Because the combination of 3D spectra offers at least two and often three independent pathways for determining sequential connectivity, the new assignment procedure is easily automated. Complete assignments are reported for the proton, carbon, and nitrogen backbone resonances of calmodulin, complexed with calcium.     

Post-uptake metabolism affects quantification of amino acid uptake

? The quantitative significance of amino acids to plant nutrition remains controversial. This experiment determined whether post-uptake metabolism and root to shoot export differ between glycine and glutamine, and examined implications for estimation of amino acid uptake. ? Field soil containing a Eucalyptus pauciflora seedling was injected with uniformly (13)C- and (15)N-labelled glycine or glutamine. I quantified (15)N and (13)C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of (12)CO(2) and (13)CO(2) from leaves and soil. ? 60-360 min after addition of amino acid, intact molecules of U-(13)C,(15)N glutamine were < 5% of (15)N excess in roots, whereas U-(13)C,(15)N glycine was 30-100% of (15)N excess in roots. Intact molecules of glutamine, but not glycine, were exported from roots to shoots. ? Post-uptake metabolism and transport complicate interpretation of isotope labelling such that root and shoot contents of intact amino acid, (13)C and (15)N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, (13)C and (15)N by determining the turnover of amino acids within roots. Alternatively, post-uptake metabolism and transport could be minimized by harvesting plants within minutes of isotope addition.     

(H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins

Triple resonance HN(COCA)NH pulse sequences for correlating 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins that utilize overlapping coherence transfer periods provide increased sensitivityrelative to pulse sequences that utilize sequential coherence transfer periods. Although theoverlapping sequence elements reduce the overall duration of the pulse sequences, theprincipal benefit derives from a reduction in the number of 180° pulses. Two versions of thetechnique are presented: a 3D (H)N(COCA)NH experiment that correlates 15N(i),1H(i-1), and 15N(i-1) spins, and a 3D HN(COCA)NH experiment that correlates 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins by simultaneously encoding the 1H(i) and 15N(i) chemical shiftsduring the t1 evolution period. The methods are demonstrated on a 13C/15N-enriched sampleof the protein ubiquitin and are easily adapted for application to 2H/13C/15N-enrichedproteins.     

Carbonyl carbon transverse relaxation dispersion measurements and ms-μs timescale motion in a protein hydrogen bond network

A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R(2), dispersion experiment for carbonyl carbons was designed and executed to detect micros-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C(alpha)-C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly (13)C enriched proteins, unless care is taken to minimize the perturbation of the C(alpha) magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the C(alpha) region of the spectrum) pulses were employed in order to minimize C' signal modulation by C(alpha)-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [(13)C,(15)N, (2)H] ((2)H at non-labile hydrogen sites) labeled, and (2) uniformly (15)N/selectively-(13)C' labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly (13)C/(15)N/(2)H labeled sample was well suited to measure (15)N and (1)H R(2) dispersion as well as (13)C' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.     

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.