Measurement Of Scalar Couplings Research Articles

5-Formylcytosine weakens the G-C pair and imparts local conformational fluctuations to DNA duplexes.

DNA epigenetic modifications such as 5-methyl (5mC), 5-hydroxymethyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine have unique and specific biological roles. Crystallographic studies of 5mC containing duplexes were conducted in the A-, B- or the intermediate E-DNA polymorphic forms. 5fC-modified duplexes initially observed in the disputed F-DNA architecture were subsequently crystallized in the A-form, suggesting that epigenetic modifications enable DNA sequences to adopt diverse conformational states that plausibly contribute to their function. Solution-state studies of these modifications were found in the B-DNA form, with marked differences in the conformational flexibility of 5fC containing duplexes in comparison to C/5mC containing duplexes, compromising the DNA duplex's stability. Herein, we systematically evaluate sensitive and commonly inaccessible NMR parameters to map the subtle differences between C, 5mC, and their oxidized (5hmC/5fC) counterparts. We observe that 15N/1H chemical shifts effectively report on the weakening of 5fC-G Watson-Crick base-pair H-bonding, extending the instability beyond any achievable within the sequence-specific changes in DNA. Triple 5fC containing sequences propagate the destabilization farther from the site of modifications, explaining reduced duplex stability upon multiple modifications. Additionally, scalar and residual dipolar coupling measurements unravel local sugar pucker fluctuations. One-bond 13C-1H scalar coupling measurements point towards a significant deviation away from the anticipated C2'-endo pucker for the 5fC modified nucleotide. Structural models obtained employing 13C-1H residual dipolar couplings and inter-proton distances corroborate the sugar pucker's deviation for 5fC modified DNA duplexes. The changes in the sugar pucker equilibria remain local to the 5fC modified nucleotide sans additive/long-range effects arising from multiple contiguous modifications. These observations highlight the impact of a major groove modification that alters the physical properties of DNA duplex without disturbing the Watson-Crick face. The changes observed in our studies for the 5fC containing DNA contrast with the perturbations induced by damage/lesion highlight the varied conformational preferences that modified nucleobases impart to the DNA duplex. As sequence-specific DNA transactions are rooted in the base-pair stability and pucker deviations, the observed structural perturbations for 5fC-modified DNA potentially play critical functional roles, such as protein-DNA recognition and interactions.

G-SERF Editing in Two-Dimensional Pure-Shift Total Correlation Spectroscopy: Scalar Coupling Measurements for a Group of Spins in Organic Molecules.

A novel G-SERF-PSYCHE-TOCSY (gradient encoded selective refocusing in pure shift yielded by chirp excitation version of total correlation spectroscopy) NMR pulse scheme has been proposed, which produces TOCSY chemical shift correlations, on one hand, and scalar coupling values for the spins scalarly coupled to irradiated resonances, by showing them as doublets along the indirect dimension, on the other. Therefore, recording such an experiment, for a group of spins with overlapping chemical shifts, in organic molecules can adequately provide scalar coupling information in a G-SERF manner along the indirect dimensions, and they can be assigned to particular spin pairs. Such COSY chemical shift correlations (which appear as doublets for the scalarly coupled spins) can be readily discriminated from the TOCSY peaks (which do not show such splitting) in the G-SERF-PSYCHE-TOCSY spectrum.

Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB.

Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.

Inferring Structural Ensembles from Noisy Experiments and Molecular Dynamics: Correcting Force Field Bias with Bayesian Energy Landscape Tilting

Inferring biomolecular conformation from experiment is a fundamental goal of structural biology. Structure determination often requires the combination of modeling and experiment, but the vast majority of approaches model only a single conformation, provide limited uncertainty information, and inherit biases from assumed force fields when data are limited. Building on recent conceptual advances, we hypothesized that these biases and missing uncertainty estimates could be addressed through Bayesian Energy Landscape Tilting (BELT), a scheme that enables the systematic computation of fully Bayesian hyperensembles over conformational ensembles. As a test of BELT's ability to correct force field bias, we show that conformational ensembles of trialanine derived from five different force fields (ff96, ff99, ff99sbnmr-ildn, CHARMM27, and OPLS-AA) and chemical shift and scalar coupling measurements give convergent values of the peptide's α, β, and PPII conformational populations. Furthermore, the ensembles recover set-aside measurements not used in the fitting. BELT's principled combination of simulation and limited experimental data promises rigorous assessment of force field bias and sets a foundation for modeling ensembles and uncertainties in complex biomolecular systems.

Molecular Basis for the Association of Human E4B U Box Ubiquitin Ligase with E2-Conjugating Enzymes UbcH5c and Ubc4

Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.

Synthesis and NMR Spectroscopic Characterization of Organometallics in the Laboratory of Wolfgang von Philipsborn: Reminiscences of Former Graduate Students

On the occasion of his 80th birthday, former graduate students from the group of Prof. Wolfgang von Philipsborn from the Institute of Organic Chemistry, University of Zurich, describe from a personal perspective the scientific achievements of his group in the field of nuclear magnetic resonance, mainly applied to transition metals and in the field of organometallic chemistry. Interest in metal chemical shifts is driven by the need to understand reactivity of organometallic compounds in catalysis. Progress in the field is very much related to the technical development of NMR instruments. The range of experiments spans from simple 1D experiments with direct metal detection to 2D NMR experiments, in which metal frequencies are encoded via their attached protons or phosphorous ligands. Other examples come from the structural biology of metal-containing proteins or form the measurement of scalar couplings to quadrupolar nuclei via lineshape analysis. A particular emphasis is presented on how collaborations from various groups at the campus have been fruitful to the scientific progress in the von Philipsborn group. The article also contains a number of personal anecdotes that document life of the graduate students in his group at that time.

Combined NMR three‐bond scalar coupling measurements and QM calculations to calculate OH‐rotamer equilibrium of polyalcohols

A combined but independently applied NMR and QM procedure has been used to investigate the conformational properties of the exchangeable hydroxyl protons of polyalcohols. In this study, to demonstrate the applicability of such a strategy, we investigated a simple monosaccharide, i.e. alpha- and beta-anomers of a D-glucopyranoside derivative. The redundant set of experimental vicinal homonuclear and heteronuclear scalar couplings involving the OH-protons obtained for both anomers of our model compound were simultaneously analyzed to yield the preferred OH-rotamer populations and moreover to parametrize a new Karplus-type equation for (3)J(C(i-1)OH(i)) coupling. The populations of the lowest energy conformers and the conformational-averaged coupling constants were independently calculated using the QM approach in both vacuum and chloroform. The similarity of the estimated rotamer populations obtained by two very different techniques and the similarity of the experimental and calculated coupling constants suggest that these approaches can be used in conjunction and in a fully integrated way to determine a more accurate atomic level description of molecular conformers.

Magnetic Resonance Spectroscopy with Longitudinal Multispin Orders

Longitudinal multispin orders can be created in spin systems that exhibit scalar, dipolar or quadrupolar couplings. They provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. They cannot be separated by phase cycling or gradient selection methods which are the only known modes of separating different coherences. In this review we describe the frequency cycling procedure for separating various orders in weakly and strongly coupled spin systems. We provide the analytical solutions that permit determination of the frequency cycle for different spin systems. We also discuss the creation of longitudinal orders through relaxation. Finally we highlight the potential applications including spectral editing, measurement of relative signs of scalar couplings and structural properties of molecules.

Limits on Variations in Protein Backbone Dynamics from Precise Measurements of Scalar Couplings

3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' couplings, all related to the backbone torsion angle phi, were measured for the third immunoglobulin binding domain of protein G, or GB3. Measurements were carried out using both previously published methods and novel sequences based on the multiple-quantum principle, which limit attenuation of experimental couplings caused by finite lifetimes of the spin states of passive spins. High reproducibility between the multiple-quantum and conventional approaches confirms the accuracy of the measurements. With few exceptions, close agreement between 3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' and values predicted by their respective Karplus equations is observed. For the three types of couplings, up to 20% better agreement is obtained when fitting the experimental couplings to a dynamic ensemble NMR structure, which has a phi angle root-mean-square spread of 9 +/- 4 degrees and was previously calculated on the basis of a very extensive set of residual dipolar couplings, than for any single static NMR structure. Fits of 3J couplings to a 1.1-A X-ray structure, with hydrogens added in idealized positions, are 40-90% worse. Approximately half of the improvement when fitting to the NMR structures relates to the amide proton deviating from its idealized, in-peptide-plane position, indicating that the positioning of hydrogens relative to the backbone atoms is one of the factors limiting the accuracy at which the backbone torsion angle phi can be extracted from 3J couplings. Introducing an additional, residue-specific variable for the amplitude of phi angle fluctuations does not yield a statistically significant improvement when fitting to a set of dynamic Karplus curves, pointing to a homogeneous behavior of these amplitudes.

NMR measurements of scalar-coupling distributions in disordered solids

The measurement of scalar (J) couplings by solid-state NMR is a field of great interest, since this interaction is a rich source of local structural information, complementary to dipolar and chemical shift interactions. Here, we first demonstrate that J-coupling distributions exist and can be observed in disordered solids, as illustrated with the observation of a pair-specific distribution of (2)J((31)P-N-(31)P) couplings in a bis-phosphino amine, and we investigate the potential effects of such distributions on the measurement of average J-coupling constants. Second, we show that the measurement of two-dimensional (2D) distributions of J-couplings provides a much richer probe of local structural disorder than one-dimensional distributions, and we introduce new methods that provide different (selective or non-selective) ways of measuring 2D J distributions in a wide range of disordered systems. These methods are finally applied to a slightly disordered polymorphic sample of fully (13)C-enriched cellulose, and then to the bis-phosphino amine sample, from which 2D (2)J(PP)-coupling distributions are clearly identified and interpreted.

Influence of the 2′-Hydroxyl Group Conformation on the Stability of A-form Helices in RNA

The 2'-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2'-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution. Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2'-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3' or towards the base, where the 2'-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k(ex) rate of the 2'-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2'-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure. The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2'-OH group with both the structure and the function of RNA and RNA-ligand complexes.

Bayesian estimation of Karplus parameters and torsion angles from three-bond scalar couplings constants

We apply Bayesian inference to analyze three-bond scalar coupling constants in an objective and consistent way. The Karplus curve and a Gaussian error law are used to model scalar coupling measurements. By applying Bayes’ theorem, we obtain a probability distribution for all unknowns, i.e., the torsion angles, the Karplus parameters, and the standard deviation of the Gaussian. We infer all these unknowns from scalar coupling data using Markov chain Monte Carlo sampling and analytically derive a probability distribution that only involves the torsion angles.

Analysis of longitudinal multispin orders in strongly coupled spin systems

Longitudinal multispin orders provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. Analysis of longitudinal orders has been made in strongly coupled AB and ABX spin systems to determine the dependence of strong coupling parameter on these orders. Experimental and simulated spectra of various longitudinal orders are illustrated for these spin systems. This general procedure can be extended to broad range of spin systems to understand the influence of strong coupling on longitudinal orders.

Correlation of the Sweetness of Variants of the Protein Brazzein with Patterns of Hydrogen Bonds Detected by NMR Spectroscopy

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (trans-hydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone 1HN, 15N, and 13C' atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the alpha-helix, where it is kinked, and one hydrogen bond in the middle of beta-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.

Direct measurement of angles between bond vectors in high-resolution NMR.

Angles between two interatomic vectors are measured for structure elucidation in solution nuclear magnetic resonance (NMR). The angles can be determined directly by using the effects of dipole-dipole cross-correlated relaxation of double-quantum and zero-quantum coherences. The measured rates can be directly related to the angular geometry without need for calibration of a Karplus-type curve, as is the case for scalar coupling measurements, and depend only on the rotational correlation time of the molecule as an empirical parameter. This makes the determination of torsional angles independent from the measurement of coupling constants. The two interatomic vectors can in principle be arbitrarily far apart. The method was demonstrated on the measurement of the peptide backbone angle psi in the protein rhodniin, which is difficult to determine in solution by NMR spectroscopy.

Modern multi-dimensional protein NMR spectroscopy: II. New experimental approaches

The first part of this review, published in the previous issue of Trends in Analytical Chemistry, dealt mainly with the new heteronuclear three-dimensional and four-dimensional NMR methods for the assignment of 1H, 13C and 15N resonances in larger proteins. In this part we will describe three recent developments in NMR techniques that still have to see their full impact in practice: (a) heteronuclear cross polarization as an alternative to INEPT for heteronuclear coherence transfer, (b) principles of the measurement of scalar couplings that are (much) smaller than the resonance linewidths, and (c) the application of pulsed field gradients in high-resolution NMR.

Separation of Longitudinal Multispin Order by Frequency Cycling

Longitudinal multispin orders provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. Analysis of longitudinal orders has been made in strongly coupled AB and ABX spin systems to determine the dependence of strong coupling parameter on these orders. Experimental and simulated spectra of various longitudinal orders are illustrated for these spin systems. This general procedure can be extended to broad range of spin systems to understand the influence of strong coupling on longitudinal orders.

Structure of an unusually stable RNA hairpin

The structure of a very common RNA hairpin, 5'GGAC(UUCG)GUCC, has been determined in solution by NMR spectroscopy. The loop sequence, UUCG, occurs exceptionally often in ribosomal and other RNAs, and may serve as a nucleation site for RNA folding and as a protein recognition site. Reverse transcriptase cannot read through this loop, although it normally transcribes RNA secondary structure motifs. A hairpin with that loop displays unusually high thermodynamic stability; its stability decreases when conserved nucleotides are mutated. The three-dimensional structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR using distance geometry, followed by restrained energy minimization. The structure was well-defined despite the conservative use of interproton distances, by constraining the backbone conformation by means of scalar coupling measurements. A mismatch G.U base pair, with syn-guanosine, closes the stem. This hairpin has a loop of only two nucleotides; both adopt C2'-endo sugar pucker. A sharp turn in the phosphodiester backbone is stabilized by a specific cytosine-phosphate contact, probably a hydrogen bond, and by stacking of the cytosine nucleotide on the G.U base pair. The structural features of the loop can explain the unusual thermodynamic stability of this hairpin and its sensitivity to mutations of loop nucleotides.

ChemInform Abstract: Generation of Pure Phase NMR Subspectra for Measurement of Homonuclear Coupling Constants

Recently it has been demonstrated that dramatic spectral simplification can be obtained by editing a regular ‘H NMR spectrum into a set of subspectra by means of the one-dimensional HOHAHA experiment (I). As pointed out earlier, scalar couplings are difficult to measure from such a spectrum because of phase and intensity distortions of the muhiplet components in the various subspectra. Here, it is demonstrated that pure phase subspectra can be obtained readily by combination of the 1D HOHAHA experiment with the z-filtering technique (2). Guidelines for minimizing the number of T averages, needed for the z filter, are also presented. If the Zeeman term from the Hamiltonian is eliminated, the spin system will evolve solely under the influence of scalar couplings. Braunschweiler and Ernst (3) lirst utilized this concept in isotropic liquids for generating total coherence transfer (TOCSY) 2D spectra of spin systems of coupled protons. A number of more efficient mixing schemes based on the same mechanism have since been reported (1, 4, 5). We prefer to use the so-called MLEV- 17 sequence (4) for mixing because it covers a substantial bandwidth (kO.3~) and because it is relatively insensitive to exact phase shifting or pulse width adjustment. Moreover, this mixing scheme lengthens the apparent Tip (4, 5) so that less signal is lost during this mixing period relative to other mixing schemes. To record pure phase spectra, Braunschweiler and Ernst proposed cc&ding a number of spectra, recorded with different mixing times. This procedure works satisfactorily when few spins are involved in a J-coupled network. For larger spin systems however, this procedure is not perfect because the magnetization transfer appears to lose its oscillatory character, i.e., the oscillation is overdamped. Consequently, the spectra obtained are not 100% absorptive and measurement of scalar couplings may lead to errors. Moreover, in a large network of coupled spins (alkaloids, steroids, etc.) averaging over a number of mixing times, some of which would have to be quite long, gives a subspectrum of nearly the entire molecule and therefore defeats the purpose of the experiment. As demonstrated here, the z-filtering technique, proposed by Slarensen

Generation of pure phase NMR subspectra for measurement of homonuclear coupling constants

Recently it has been demonstrated that dramatic spectral simplification can be obtained by editing a regular ‘H NMR spectrum into a set of subspectra by means of the one-dimensional HOHAHA experiment (I). As pointed out earlier, scalar couplings are difficult to measure from such a spectrum because of phase and intensity distortions of the muhiplet components in the various subspectra. Here, it is demonstrated that pure phase subspectra can be obtained readily by combination of the 1D HOHAHA experiment with the z-filtering technique (2). Guidelines for minimizing the number of T averages, needed for the z filter, are also presented. If the Zeeman term from the Hamiltonian is eliminated, the spin system will evolve solely under the influence of scalar couplings. Braunschweiler and Ernst (3) lirst utilized this concept in isotropic liquids for generating total coherence transfer (TOCSY) 2D spectra of spin systems of coupled protons. A number of more efficient mixing schemes based on the same mechanism have since been reported (1, 4, 5). We prefer to use the so-called MLEV- 17 sequence (4) for mixing because it covers a substantial bandwidth (kO.3~) and because it is relatively insensitive to exact phase shifting or pulse width adjustment. Moreover, this mixing scheme lengthens the apparent Tip (4, 5) so that less signal is lost during this mixing period relative to other mixing schemes. To record pure phase spectra, Braunschweiler and Ernst proposed cc&ding a number of spectra, recorded with different mixing times. This procedure works satisfactorily when few spins are involved in a J-coupled network. For larger spin systems however, this procedure is not perfect because the magnetization transfer appears to lose its oscillatory character, i.e., the oscillation is overdamped. Consequently, the spectra obtained are not 100% absorptive and measurement of scalar couplings may lead to errors. Moreover, in a large network of coupled spins (alkaloids, steroids, etc.) averaging over a number of mixing times, some of which would have to be quite long, gives a subspectrum of nearly the entire molecule and therefore defeats the purpose of the experiment. As demonstrated here, the z-filtering technique, proposed by Slarensen