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.     

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.     

Physical interpretation of residual dipolar couplings in neutral aligned media

A novel method is described for rapidly calculating alignment tensors from hydrodynamic shape, required for the prediction of residual dipolar couplings in neutral aligned media. Simulations of alignment were used to show that for steric restriction at a planar surface, the alignment process is dependent on linear hydrodynamic length. However, as discussed, previous methods are not in agreement with this observation. Therefore, the method presented here is the first to provide simple, accurate predictions of the alignment tensor for neutral and dilute media, while being consistent with simulations of alignment. It provides predictions in a fraction of the time of a simulation approach, while aiding physical intuition by providing a direct link between shape and alignment. Not only is this physically gratifying, but it also permits residual dipolar couplings to be applied in demanding situations where simulations of alignment are not desirable, such as in studies of molecular dynamics.     

Chi1 torsion angle dynamics in proteins from dipolar couplings.

Experiments are presented for the measurement of one-bond carbon-proton dipolar coupling values at CH and CH2 ositions in 13C-labeled, approximately 50% fractionally deuterated proteins. 13Cbeta-1Hbeta dipolar couplings have been measured for 38 of 49 possible residues in the 63-amino-acid B1 domain of peptostreptococcal protein L in two aligning media and interpreted in the context of side-chain chi1 torsion angle dynamics. The beta protons for 18 of the 25 beta-methylene-containing amino acids for which dipolar data are available can be unambiguously stereoassigned, and for those residues which are best fit to a single rotamer model the chi(1) angles obtained deviate from crystal structure values by only 5.2 degrees (rmsd). The results for 11 other residues are significantly better fit by a model that assumes jumps between the three canonical (chi1 approximately -60 degrees, 60 degrees, 180 degrees ) rotamers. Relative populations of the rotamers are determined to within +/-6% uncertainty on average and correlate with dihedral angles observed for the three molecules in the crystal asymmetric unit. Entropic penalties for quenching chi1 jumps are considered for six mobile residues thought to be involved in binding to human immunoglobulins. This study demonstrates that dipolar couplings may be used to characterize both the conformation of static residues and side-chain motion with high precision.     

Simultaneous determination of 1H-1H and 1H-13C residual dipolar couplings in a chiral liquid crystal solvent using a natural abundance HSQC experiment

A high-resolution, phase-sensitive, natural abundance F2-coupled 1H-13C HSQC (F2HSQC) NMR experiment was developed to measure simultaneously both (n)D(HH) and 1D(CH) residual dipolar couplings (RDCs) of small molecules present in a chiral polypeptide liquid crystal solvent system composed of poly-gamma-benzyl-L-glutamate (PBLG) in CDCl3. Because this is an indirect-detection NMR experiment, the relatively small amount of sample (7.5 mg in this study) and short acquisition times (5 h) that are required make this HSQC experiment well suited for samples that are either limited in solubility or in quantity or require short analysis times. The F2HSQC experiment can be performed without any specialized equipment or sample modification and can enhance our ability to measure RDCs accurately and rapidly in polypeptide liquid crystal solvents.     

Measurement of 1H-1H residual dipolar coupling constants for structural studies of medium size molecules.

Residual dipolar coupling constants (RDCs) are being increasingly applied to elucidate the configuration and conformation of small organic molecules, peptides and oligosaccharides. In this paper we describe a set of robust 1D NMR methods for accurate and precise measurement of proton-proton RDCs of small and medium size molecules. The performance of these techniques is not impeded by the presence of overlapping and broad (1)H multiplets that are typically observed for such molecules in weakly aligned media. The use of these techniques provides access to a large pool of proton-proton RDCs opening new avenues for the solution structure elucidation of medium size molecules by NMR. The techniques are illustrated on the determination of the alignment tensor of the reducing monosaccharide ring of cellobiose and the determination of the relative configuration of sodium cholate.     

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.     

Measurement of carbon-proton dipolar couplings in liquid crystals using DAPT

Dipolar couplings provide valuable information on order and dynamics in liquid crystals. For measuring heteronuclear dipolar couplings in oriented systems, a new separated local field experiment is presented here. The method is based on the dipolar assisted polarization transfer (DAPT) pulse sequence proposed recently (Chem. Phys. Lett. 2007, 439, 407) for transfer of polarization between two spins I and S. DAPT utilizes the evolution of magnetization of the I and S spins under two blocks of phase shifted BLEW-12 pulses on the I spin separated by a 90 degree pulse on the S spin. Compared to the rotating frame techniques based on Hartmann-Hahn match, this approach is easy to implement and is independent of any matching conditions. DAPT can be utilized either as a proton encoded local field (PELF) technique or as a separated local field (SLF) technique, which means that the heteronuclear dipolar coupling can be obtained by following either the evolution of the abundant spin like proton (PELF) or that of the rare spin such as carbon (SLF). We have demonstrated the use of DAPT both as a PELF and as a SLF technique on an oriented liquid crystalline sample at room temperature and also have compared its performance with PISEMA. We have also incorporated modifications to the original DAPT pulse sequence for (i) improving its sensitivity and (ii) removing carrier offset dependence.     

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.     

Measurement of 13C-1H dipolar couplings in solids by using ultra-fast magic-angle spinning NMR spectroscopy with symmetry-based sequences

We show that (13)C-(1)H dipolar couplings in fully protonated organic solids can be measured by applying a Symmetry-based Resonance-Echo DOuble-Resonance (S-REDOR) experiment at ultra-fast Magic-Angle Spinning (MAS). The (13)C-(1)H dipolar couplings are recovered by using the R12 recoupling scheme, while the interference of (1)H-(1)H dipolar couplings are suppressed by the symmetry properties of this sequence and the use of high MAS frequency (65 kHz). The R12 method is especially advantageous for large (13)C-(1)H dipolar interactions, since the dipolar recoupling time can be incremented by steps as short as one rotor period. This allows a fine sampling for the rising part of the dipolar dephasing curve. We demonstrate experimentally that one-bond (13)C-(1)H dipolar coupling in the order of 22 kHz can be accurately determined. Furthermore, the proposed method allows a rapid evaluation of the dipolar coupling by fitting the S-REDOR dipolar dephasing curve with an analytical expression.     

On the origin of residual dipolar couplings from denatured proteins

Effects of steric obstruction on random flight chains are examined. Spatial probability distributions are elaborated to calculate residual dipolar couplings and residual chemical shift anisotropy, parameters that are acquired by NMR spectroscopy from solutes dissolved in dilute liquid crystals. Calculations yield chain length and residue position-dependent values in good agreement with simulations to provide understanding of recently acquired data from denatured proteins.     

Bound-state residual dipolar couplings for rapidly exchanging ligands of His-tagged proteins

The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound-state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.     

Refinement of local and long-range structural order in theophylline-binding RNA using (13)C-(1)H residual dipolar couplings and restrained molecular dynamics.

13C-(1)H residual dipolar couplings (RDC) have been measured for the bases and sugars in the theophylline-binding RNA aptamer, dissolved in filamentous phage medium, and used to investigate the long-range structural and dynamic behavior of the molecule in the solution state. The orientation dependent RDC provide additional restraints to further refine the overall structure of the RNA-theophylline complex, whose long-range order was poorly defined in the NOE-based structural ensemble. Structure refinement using RDC normally assumes that molecular alignment can be characterized by a single tensor and that the molecule is essentially rigid. To address the validity of this assumption for the complex of interest, we have analyzed distinct domains of the RNA molecule separately, so that local structure and alignment tensors experienced by each region are independently determined. Alignment tensors for the stem regions of the molecule were allowed to float freely during a restrained molecular dynamics structure refinement protocol and found to converge to similar magnitudes. During the second stage of the calculation, a single alignment tensor was thus applied for the whole molecule and an average molecular conformation satisfying all experimental data was determined. Semirigid-body molecular dynamics calculations were used to reorient the refined helical regions to a relative orientation consistent with this alignment tensor, allowing determination of the global conformation of the molecule. Simultaneously, the local structure of the theophylline-binding core of the molecule was refined under the influence of this common tensor. The final ensemble has an average pairwise root mean square deviation of 1.50 +/- 0.19 A taken over all heavy atoms, compared to 3.5 +/- 1.1 A for the ensemble determined without residual dipolar coupling. This study illustrates the importance of considering both the local and long-range nature of RDC when applying these restraints to structure refinements of nucleic acids.     

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.     

Precision 1H-1H distance measurement via 13C NMR signals: utilization of 1H-1H double-quantum dipolar interactions recoupled under magic angle spinning conditions

We applied the POST-C7 DQ-dipolar recoupling pulse sequence to the measurement of (1)H-(1)H distances with high precision. The spectral resolution is enhanced by detecting the (1)H magnetization via (13)C signals. A least-squares fitting of the build-up curve of the transferred magnetization to the exact numerical simulations yielded a (1)H(alpha)-(1)H(beta) distance of 248 +/- 4 pm for fully (13)C-labeled L-valine. This distance agrees with the neutron diffraction study. The negative transferred magnetization clearly indicates that the direct DQ (1)H-(1)H dipolar couplings have the largest effect. The signal for the magnetization transfer builds up rapidly by the direct (1)H-(1)H dipolar coupling, and decreases to zero at longer mixing time when the relayed magnetization transfer becomes significant. This large intensity change of the signal leads to the high precision in the distance measurement. We inspected factors that limit the effective bandwidth of the POST-C7 recoupling for the (1)H and (13)C homonuclear spin systems. The spin interactions at times shorter than the cycle time of the C7 sequence were also evaluated to measure the distances. The carbon-detected 2D (1)H DQ mixing experiment was demonstrated for the measurement of multiple (1)H-(1)H distances.