Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments

NMR spectra of molecules oriented in liquid crystals provide homo- and heteronuclear dipolar couplings and thereby the geometry of the molecules. Several inequivalent dilute spins such as 13C and 15N coupled to protons form different coupled spin systems in their natural abundance and appear as satellites in the proton spectra. Identification of transitions belonging to each spin system is essential to determine heteronuclear dipolar couplings, which is a formidable task. In the present study, using 15N-1H and 13C-1H HSQC, and HMQC experiments we have selectively detected spectra of each rare spin coupled to protons. The 15N-1H and 13C-1H dipolar couplings have been determined in the natural abundance of 13C and 15N for the molecules pyrazine, pyrimidine and pyridazine oriented in a thermotropic liquid crystal.     

High-accuracy residual 1HN-13C and 1HN-1HN dipolar couplings in perdeuterated proteins

Truncation by the presence of many short-range residual dipolar couplings (RDCs) hinders the observation of long-range RDCs in weakly aligned biomacromolecules. Perdeuteration of proteins followed by reprotonation of labile hydrogen positions greatly alleviates this problem. Here we show that for small perdeuterated proteins, a large number (up to 10 in protein G) of long-range RDCs to 13C and 1HN can be observed from individual amide protons. The 1HN <--> 13C RDCs comprise correlations to 13Calpha, 13Cbeta, and 13C' nuclei of the same and the preceding amino acid, as well as 13C' nuclei of hydrogen-bonded amino acids. The accuracy of the coupling constants is very high and defines individual internuclear distances to within few picometers. Deviations between measured RDC values and values predicted from the 1.1 A crystal structure of protein G are mainly found in two surface-exposed loop regions. The deviations show a strong correlation to the B-factor of the crystal structure.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

Measurements of side-chain 13C-13C residual dipolar couplings in uniformly deuterated proteins

13C-only spectroscopy was used to measure multiple residual (13)C-(13)C dipolar couplings (RDCs) in uniformly deuterated and (13)C-labeled proteins. We demonstrate that (13)C-start and (13)C-observe spectra can be routinely used to measure an extensive set of the side-chain residual (13)C-(13)C dipolar couplings upon partial alignment of human ubiquitin in the presence of bacteriophages Pf1. We establish that, among different broadband polarization transfer schemes, the FLOPSY family can be used to exchange magnetization between a J coupled network of spins while largely decoupling dipolar interactions between these spins. An excellent correlation between measured RDCs and the 3D structure of the protein was observed, indicating a potential use of the (13)C-(13)C RDCs in the structure determination of perdeuterated proteins.     

Selective measurement of heteronuclear 1H-13C dipolar couplings in motionally heterogeneous semicrystalline polymer systems

A pulse sequence for the selective recoupling of heteronuclear dipolar interactions in mobile amorphous phase of powdered semicrystalline polymers is described. 1H-13C dipolar interactions are selectively measured by PISEMA-type sequence. Selection of 13C magnetization originating from amorphous phase is achieved by a train of saturation pulses followed by a short delay and a direct excitation pulse on 13C spins. The development of undesired net 13C magnetization during the recoupling sequence is prevented by the efficient "reverse" 13C --> 1H cross-polarization. The efficacy of the 2D method to measure 1H-13C dipolar couplings selectively for mobile components is demonstrated on powdered crystalline L-alanine, semicrystalline polyethylene, and nanocomposite polyamide-6/montmorillonite.     

Molecular structure extracted from residual dipolar couplings: diphenylmethane dissolved in a nematic liquid crystal

This paper describes an analysis of 1H-1H residual dipolar couplings (RDCs) in diphenylmethane (DPM) dissolved in a nematic liquid crystal, reported by Celebre et al. [J. Chem. Phys. 118, 6417 (2003)]. In that article, the conformational distribution function for DPM was extracted from the RDCs, using the additive potential (AP) model which is based on the molecular-field theory. The AP approach is a powerful, and frequently used, tool for analysis of the nuclear-magnetic-resonance (NMR) parameters in liquid crystals. It requires, however, a priori knowledge of the functional form of the torsional potential, which may even for a simple molecule, such as DPM, be complicated to determine. Here, we analyze the same set of the RDCs using our APME procedure, which is a hybrid model based on the AP approach and maximum entropy (ME) theory. The APME procedure does not require any assumptions about the functional form of the torsional potential and, in contrast with the ME method, is applicable to weakly ordered systems. In the investigation reported in the present study, the results from the APME analysis are in good agreement with the AP interpretation, whereas the ME approach essentially fails in the extraction of the conformational distribution function for DPM.     

1H-1H dipolar couplings provide a unique probe of RNA backbone structure

A NMR method is described that permits simultaneous measurement of the geminal 2JH1H2 + 2DH1H2 splitting and the sum of the 1JCH1 + 1DCH1 + 1JCH2 + 1DCH2 couplings for methylene groups, where 2DH1H2 and 1DCH are residual dipolar couplings, occurring when molecules are weakly oriented relative to the magnetic field. By suppressing either the upfield or downfield half of the 1H-1H geminal doublet, the experiment yields improved resolution relative to regular two-dimensional 1H-13C correlation spectra, making it applicable to systems of considerable complexity. The method is demonstrated for measurement of all 2DH5'H5' couplings in a 24-nucleotide 13C-enriched RNA stem loop structure, weakly aligned in liquid crystalline Pf1. The method is equally applicable to methylene groups in 13C-labeled proteins and to natural abundance samples of smaller molecules.     

13C direct detected NMR increases the detectability of residual dipolar couplings

13C direct detection is becoming an increasingly efficient approach to identify signals of residues that escape detection in 1H detected experiments. Pulse sequences have been developed to obtain 1H partially recoupled experiments for the measurement of the 1JHalphaCalpha and 1JHN couplings with the same resolution available in conventional 1H detected experiments. A consistent set of backbone rdc obtained without any 1H-based experiment has been obtained and shown to be effective for protein solution structure determination.     

Measurement of long-range 1H-1H dipolar couplings in weakly aligned proteins

Measurement of 1H-1H dipolar couplings in macromolecules, weakly oriented by a dilute liquid crystalline medium, is generally limited to the largest such interactions. By removing dipolar couplings to nearest neighbors, either by decoupling, deuteration, or both, more remote interactions become accessible. The approach is demonstrated for measurement of amide-amide interactions in the proteins calmodulin and ubiquitin and permits observation of direct dipolar couplings between protons up to 7 A apart. Quantitative evaluation of 1H-1H dipolar couplings measured in ubiquitin shows excellent agreement with its solution structure.     

Empirical determination of the absolute configuration of small chiral molecules using natural abundance 2H NMR in chiral liquid crystals

Natural abundance deuterium 2D NMR spectroscopy in polypeptide liquid crystals is used for empirically determining the absolute configuration of small chiral molecules.     

Real-time monitoring of a dynamic molecular system using 1H-13C HSQC NMR spectroscopy with an optimized 13C window

The kinetic and thermodynamic parameters of an equilibrating network involving 8 molecules can be determined from a series of quick and highly resolved (1)H-(13)C HSQC NMR experiments obtained using a reduced carbon spectral window.     

De novo determination of protein backbone structure from residual dipolar couplings using Rosetta

As genome-sequencing projects rapidly increase the database of protein sequences, the gap between known sequences and known structures continues to grow exponentially, increasing the demand to accelerate structure determination methods. Residual dipolar couplings (RDCs) are an attractive source of experimental restraints for NMR structure determination, particularly rapid, high-throughput methods, because they yield both local and long-range orientational information and can be easily measured and assigned once the backbone resonances of a protein have been assigned. While very extensive RDC data sets have been used to determine the structure of ubiquitin, it is unclear to what extent such methods will generalize to larger proteins with less complete data sets. Here we incorporate experimental RDC restraints into Rosetta, an ab initio structure prediction method, and demonstrate that the combined algorithm provides a general method for de novo determination of a variety of protein folds from RDC data. Backbone structures for multiple proteins up to approximately 125 residues in length and spanning a range of topological complexities are rapidly and reproducibly generated using data sets that are insufficient in isolation to uniquely determine the protein fold de novo, although ambiguities and errors are observed for proteins with symmetry about an axis of the alignment tensor. The models generated are not high-resolution structures completely defined by experimental data but are sufficiently accurate to accelerate traditional high-resolution NMR structure determination and provide structure-based functional insights.     

Conformational analysis of a flexible oligosaccharide using residual dipolar couplings.

We present a new approach to the analysis of the conformational and the motional properties of an oligosaccharide, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside. The approach relies on an order matrix analysis of residual dipolar couplings in the solution state. By combining a number of different types of couplings, (1)D(CH), (2)D(CH), and D(HH), an order matrix is solved for each ring of the trimannoside. The resulting order parameters indicate the internal motion at the alpha (1,3) linkage to be limited, while significant motion is suggested at the alpha (1,6) linkage. Two structures for the trimannoside were determined by aligning the order tensor principal axes obtained from two different orienting media, bicelles and phage. The very similar conformations at the alpha (1,3) linkage of these two structures confirm that the internal motion at the alpha (1,3) linkage is small and the conformation is a good representation of a single preferred structure. The different conformations at the alpha (1,6) linkage suggest that the motional amplitudes are large and the conformations must be viewed as virtual conformers. Compared with traditional NMR methods, data acquisition is easy and data analysis is straightforward.     

Protein Backbone 1H(N)-13Calpha and 15N-13Calpha residual dipolar and J couplings: new constraints for NMR structure determination

A simple, sensitivity-enhanced experiment was devised for accurate measurement of backbone 15N-13Calpha and 1HN-13Calpha couplings in proteins. The measured residual dipolar couplings 2DHCA, 1DNCA, 3DHCA, and 2DNCA for protein GB1 display very good agreement with the refined NMR structure (PDB code: 3GB1). A Karplus-type relationship between the one-bond 1JNCA couplings and the backbone dihedral psi angles holds, and on the basis of the two-bond 2JNCA couplings a secondary structure index can be established.     

Reducing the computational cost of NMR crystallography of organic powders at natural isotopic abundance with the help of 13C-13C dipolar couplings

Structure determination of functional organic compounds remains a formidable challenge when the sample exists as a powder. Nuclear magnetic resonance crystallography approaches based on the comparison of experimental and Density Functional Theory (DFT)-computed ¹H chemical shifts have already demonstrated great potential for structure determination of organic powders, but limitations still persist. In this study, we discuss the possibility of using ¹³C-¹³C dipolar couplings quantified on powdered theophylline at natural isotopic abundance with the help of dynamic nuclear polarization, to realize a DFT-free, rapid screening of a pool of structures predicted by ab initio random structure search. We show that although ¹³C-¹³C dipolar couplings can identify structures possessing long range structural motifs and unit cell parameters close to those of the true structure, it must be complemented with other data to recover information about the presence and the chemical nature of the supramolecular interactions.     

Structure of methylenecyclopropane in a liquid crystal by 1H and 13C NMR

The ¹H and ¹³C NMR spectra of methylenecyclopropane, both in the isotropic phase and in the Merck IV nematic phase, were analysed. The relative signs of the ¹³C H scalar coupling constants were determined. The complete geometry deduced from this study was compared with that from a microwave study, and the discrepancies are discussed.