Residual Dipolar Couplings Research Articles

Orientation-independent quantification of macromolecular proton fraction in tissues with suppression of residual dipolar coupling.

Quantitative magnetization transfer (MT) imaging enables noninvasive characterization of the macromolecular environment of tissues. However, recent work has highlighted that the quantification of MT parameters using saturation radiofrequency (RF) pulses exhibits orientation dependence in ordered tissue structures, potentially confounding its clinical applications. Notably, in tissues with ordered structures, such as articular cartilage and myelin, the residual dipolar coupling (RDC) effect can arise owing to incomplete averaging of dipolar-dipolar interactions of water protons. In this study, we demonstrated the confounding effect of RDC on quantitative MT imaging in ordered tissues can be suppressed by using an emerging technique known as macromolecular proton fraction mapping based on spin-lock (MPF-SL). The off-resonance spin-lock RF pulse in MPF-SL could be designed to generate a strong effective spin-lock field to suppress RDC without violating the specific absorption rate and hardware limitations in clinical scans. Furthermore, suppressing the water pool contribution in MPF-SL enabled the application of a strong effective spin-lock field without confounding effects from direct water saturation. Our findings were experimentally validated using human knee specimens and healthy human cartilage. The results demonstrated that MPF-SL exhibits lower sensitivity to tissue orientation compared with , , and saturation-pulse-based MT imaging. Consequently, MPF-SL could serve as a valuable orientation-independent technique for the quantification of MPF.

Cyclic Peptide C5aR1 Antagonist Design Using Solution Conformational Analysis Derived from Residual Dipolar Couplings

Cyclic Peptide C5aR1 Antagonist Design Using Solution Conformational Analysis Derived from Residual Dipolar Couplings

Advanced Nuclear Magnetic Resonance, Fourier Transform–Infrared, Visible-NearInfrared and X-ray Diffraction Methods Used for Characterization of Organo-Mineral Fertilizers Based on Biosolids

Biosolids from stabilized sludge present a high fertilization potential, due to their rich content of nutrients and organic matter. The intrinsic and subtle properties of such fertilizers may greatly influence the fertilization efficiency. In this sense, the utility, advantages and limitations of advanced characterization methods, for the investigation of structural and dynamic properties at the microscopic scale of slightly different formulations of fertilizers were assessed. For that, three formulas of organo-mineral fertilizers based on biosolids (V1, V2 and V3), having at least 2% N, 2% P2O5, and 2% K2O, were characterized by advanced methods, such as 1H NMR relaxometry, 1H MAS and 13C CP-MAS NMR spectroscopy, 1H double-quantum NMR and FT-IR spectroscopy. Advanced structural characterization was performed using SEM, EDX and X-ray diffraction. Four dynamical components were identified in the NMR T2 distribution showing that the rigid component has a percentage larger than 90%, which explains the broad band of NMR spectra confirmed by the distributions of many components in residual dipolar coupling as were revealed by 1H DQ-NMR measurements. SEM and EDX measurements helped the identification of components from crystalline-like X-ray diffraction patterns. To evaluate the release properties of organo-mineral fertilizers, dynamic measurements of classical electric conductivity and pH were performed by placing 0.25 g of the formulas (V1, V2 and V3) in 200 mL of distilled water. The content of N and P were quantified using specific reactants, combined with VIS-nearIR spectroscopy. Two release mechanisms were observed and characterized. It was found that V3 presents the smallest release velocity but releases the largest number of fertilizers.

Stretch-Induced Ordering of Prochiral Dimethyl Sulfoxide in Anisotropic Hydrogels Analysed by 1H and 2H Nuclear Magnetic Resonance.

Nuclear spins in small molecules dissolved in stretched hydrogels typically have population-averaged residual interactions. The nuclear magnetic resonance (NMR) spectra of these systems often show additional peaks and splittings compared with free solutions. Residual dipolar couplings (RDCs) and quadrupolar couplings (RQCs) are observed for guest1H or2H nuclear spins, respectively. Dimethyl sulfoxide (DMSO) is an exquisitely sensitive probe of such biologically relevant environments since it is prochiral and becomes effectively chiral when embedded in anisotropic gelatin-based hydrogels. Measured1H RDCs and2H RQCs were used to estimate bond order parametersover a wide range of stretching extents.At the largest extent of stretching, the2H splittings were-73.0 and-9.4 Hz, similar to those found for guest molecules in liquid crystals. Inhomogeneous line broadening of the2H resonances was related to the size of the RQC due to a spatial distribution of RQCs, which was revealed using a one-dimensional slice selective imaging experiment along the stretching direction.1H NMR spectra exhibited homogeneous line broadening, with resonance integrals that indicated concealed multiplet structure. Understanding molecular bond ordering in mechanically oriented environments provides a conceptual framework for investigating more complex systems including zeolites and those foundin vivo.

NMR residual dipolar couplings investigation in the topology of house dust mite Group V allergens

Blo t 5 is an important major allergen protein from Blomia tropicalis mites, which are prevalent in tropical and subtropical regions, including Taiwan. It is a coiled-coil triple helical bundle, but there currently is ambiguity around its structural fold and packing of the three helices. We have relied on NMR residual dipolar coupling data collected from four different alignment media to confirm that Blo t 5 has left-handed helical topology and further used that data to refine its solution structure. Earlier we had described conformational epitope for a detection monoclonal antibody by exclusive use of TROSY NMR experiments that studied Blo t 5 binding with the antibody FAB’ fragment. Here, we confirm those findings with an extensive mutagenesis and biophysical study to validate the NMR epitope mapping approach proposed by us.

Bimolecular Sandwich Aggregates of Porphyrin Nanorings.

Extended π-systems often form supramolecular aggregates, drastically changing their optical and electronic properties. However, aggregation processes can be difficult to characterize or predict. Here, we show that butadiyne-linked 8- and 12-porphyrin nanorings form stable and well-defined bimolecular aggregates with remarkably sharp NMR spectra, despite their dynamic structures and high molecular weights (12.7 to 26.0 kDa). Pyridine breaks up the aggregates into their constituent rings, which are in slow exchange with the aggregates on the NMR time scale. All the aggregates have the same general two-layer sandwich structure, as deduced from NMR spectroscopy experiments, including 1H DOSY, 1H-1H COSY, TOCSY, NOESY, and 1H-13C HSQC. This structure was confirmed by analysis of residual dipolar couplings from 13C-coupled 1H-13C HSQC experiments on one of the 12-ring aggregates. Variable-temperature NMR spectroscopy revealed an internal ring-on-ring rotation process by which two π-π stacked conformers interconvert via a staggered conformation. A slower dynamic process, involving rotation of individual porphyrin units, was also detected by exchange spectroscopy in the 8-ring aggregates, implying partial disaggregation and reassociation. Molecular dynamics simulations indicate that the 8-ring aggregates are bowl-shaped and highly fluxional, compared to the 12-ring aggregates, which are cylindrical. This work demonstrates that large π-systems can form surprisingly well-defined aggregates and may inspire the design of other noncovalent assemblies.

Enantiomer Differentiation by Interaction-Specific Prediction of Residual Dipolar Couplings in Spherical-like Molecules.

Residual Dipolar Couplings (RDCs) are averaged dipolar couplings between nuclear spins of atoms in a molecule that can be measured by nuclear magnetic resonance (NMR) spectroscopy upon partial alignment by a chiral alignment medium. The estimation of differences in alignment of enantiomers may, in principle, enable the determination of absolute configuration. Here, we use molecular dynamics (MD) simulations to mimic the alignment of chiral molecules (i.e., isopinocampheol, quinuclidin-3-ol, borneol, and camphor) to the chiral poly-γ-benzyl-L-glutamate (PBLG) polymer to predict RDCs in silico and compare calculated and experimentally measured residual dipolar couplings for the four enantiomeric pairs. The aim is to validate the computational scheme for the prediction of RDCs in chiral molecules and understand the interaction leading to the alignment in more detail. We determine the indispensable importance of hydrogen bonds between a chiral molecule and the alignment medium on the overall quality of the simulated alignment and interaction poses toward high agreement with experiments. A good correlation with experimental data is found for camphor and isopinocampheol, while the correlation for quinuclidin-3-ol and borneol is lower. We attribute this observation to the high difficulty of the RDC prediction for rather almost spherical molecules. The study reveals that the prediction of alignment with small enantiomeric differences is possible with an MD-based approach; however, extended simulation times (e.g., 50-100 μs) are required to sufficiently reduce the statistical uncertainty. This may be further used for the determination of the relative, as well as absolute, configuration of chiral molecules.

Molecular structure refinement based on residual dipolar couplings using magnetic-field rotational sampling.

A method for structure refinement of molecules based on residual dipolar coupling (RDC) data is proposed. It calculates RDC values using magnetic-field rotational sampling of the rotational degrees of freedom of a molecule in conjunction with molecule-internal configurational sampling. By applying rotational sampling, as is occurring in the experiment, leading to observable RDCs, the method stays close to the experiment. It avoids the use of an alignment tensor and, therefore, the assumptions that the overall rotation of the molecule is decoupled from its internal motions and that the molecule is rigid. Two simple molecules, a relatively rigid and a very flexible cyclo-octane molecule with eight aliphatic side chains containing 24 united atoms, serve as so-called "toy model" test systems. The method demonstrates the influence of molecular flexibility, force-field dominance, and the number of RDC restraints available on the outcome of structure refinement based on RDCs. Magnetic-field rotational sampling is basically equivalent but more efficient than explicitly sampling the rotational degrees of freedom of the molecule. In addition, the performance of the method is less dependent on the number NRDC of measured RDC-values available. The restraining forces bias the overall orientation distribution of the molecule correctly. This study suggests that the information content of RDCs with respect to molecular structure is limited.

Tandem "One-Shot" Measurement of Residual Chemical Shift Anisotropy and Residual Dipolar Coupling using Biphasic Supramolecular Peptide Liquid Crystals.

Both solitary and tandem applications of residual chemical shift anisotropy (RCSA) and residual dipolar coupling (RDC) show great potential for the structural and configurational determination of organic molecules. A critical component of both RDC and RCSA methodologies is the alignment medium, whose availability is limited, especially for RCSA measurement. Moreover, reported RDC and RCSA acquisitions mainly rely on two experiments conducted under two different conditions, which are relatively time-consuming and easily cause experimental errors. Herein, a biphasic supramolecular lyotropic liquid crystalline (LLC) system was developed through the self-assembly of C21H43-CONH-V4K3-CONH2, which could act as an alignment medium for not only RDC but also RCSA extraction in DMSO-d6. Notably, the RCSA extraction was easily achieved via one-shot measurement from a single one-dimensional 13C NMR experiment, with no need for special instruments, devices, and correction. Relying on the biphasic LLC medium, meanwhile, RDC data were simply extracted from a single F1-coupled HSQC experiment, different from the standard protocol that requires two spectral acquisitions corresponding to the isotropic and anisotropic conditions. Collectively, the biphasic LLC medium is applicable for tandem RCSA and RDC measurements in one single sample, advancing the stereochemical elucidation of molecules of interest.

Molecular Structure Refinement Based on Residual Dipolar Couplings: A Comparison of the Molecular Rotational-Sampling Method with the Alignment-Tensor Approach.

In NMR experiments, residual dipolar couplings (RDCs) in a molecule can be measured by averaging the dipolar couplings (DCs) over the rotational motion of a molecule in an environment that induces a slight anisotropic orientation distribution of the molecule. Since the shape of the anisotropic distribution cannot be measured, it is standard practice to use a particular orientation distribution of the molecule with respect to the magnetic field, in the form of a so-called alignment tensor (AT), to calculate RDC-values for the molecule. Since the same alignment tensor is commonly used to calculate the different RDCs of a molecule, this approach rests on the assumption that the rotational motion of the molecule is decoupled from its internal motions and that the molecule is rigid. The validity of these two assumptions is investigated for a small, simple molecule, using a relatively rigid atomic interaction function or force field and a more flexible one. By simulating the molecule using an orientation-biasing force an anisotropic rotational distribution can be generated, for which RDCs can be obtained. Using these RDCs as target RDCs when applying one of the two approaches of structure refinement based on RDCs, it can be investigated how well the target RDCs are approximated in the RDC restraining and whether the corresponding nonuniform orientation distribution is reproduced. For the relatively rigid version of the molecule, the AT approach reproduces the target RDC-values, although the nonuniform orientation distribution of the angle θab,H between the vector r⃗ab connecting two atoms a and b in the molecule and the vector representing the direction of the magnetic field H⃗ as generated in the orientation-biasing simulation cannot be reproduced in the AT RDC-restraining simulation. For the relatively flexible version of the molecule, the AT approach fails to reproduce both the target RDC values and the nonuniform orientation distribution. For biomolecules with flexible parts, the application of the AT approach is thus not recommended. Instead, a method based on sampling of the rotational and internal degrees of freedom of the molecule should be applied in molecular structure determination or refinement based on measured RDCs.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Hydrogen bond formation may enhance RDC-based discrimination of enantiomers.

The distinction of enantiomers based on residual anisotropic parameters obtained by alignment in chiral poly-γ-benzyl-L-glutamate (PBLG) is among the strongest in high-resolution NMR spectroscopy. However, large variations in enantiodifferentiation among different solutes are frequently observed. One hypothesis is that the formation of hydrogen bonds between solute and PBLG is important for the distinction of enantiomers. With a small set of three almost spherical enantiomeric pairs, for which 1DCH residual dipolar couplings are measured, we address this issue in a systematic way: borneol contains a single functional group that can act as a hydrogen bond donor, camphor has a single group that may act as a hydrogen bond acceptor, and quinuclidinol can act as both hydrogen bond donor and acceptor. The results are unambiguous: although camphor shows low enantiodifferentiation with PBLG and alignment that can be predicted well by the purely steric TRAMITE approach, the distinction of enantiomers for the other enantiomeric pairs is significantly higher with alignment properties that must involve a specific interaction in addition to steric alignment.

Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Improved analysis of NMR chemical shift perturbations through an error estimation method

In solution NMR, chemical shift perturbation (CSP) experiments are widely employed to study intermolecular interactions. However, excluding the nonsignificant peak shift is difficult because little is known about errors in CSP. Here, to address this issue, we introduce a method for estimating errors in CSP based on the noise level. First, we developed a technique that involves line shape fitting to estimate errors in peak position via Monte Carlo simulations. Second, this technique was applied to estimate errors in CSP. In intermolecular interaction analysis of VAP-A with SNX2, error estimation of CSP enabled the evaluation of small but significant changes in peak position and yielded detailed insights that are unattainable with conventional CSP analysis. Third, this technique was successfully applied to estimate errors in residual dipolar couplings. In conclusion, our error estimation method improves CSP analysis by excluding the nonsignificant peak shift.

Full configurational and conformational analysis of artemisinin by one-bond carbon-carbon residual dipolar couplings at natural abundance.

Configurational and conformational analysis of the biologically relevant natural product artemisinin was conducted using carbon-carbon residual dipolar couplings (1DCC RDCs) at natural abundance. These RDCs were measured through the 2D-INADEQUATE NMR experiment using a sample aligned in a compressed poly (methyl methacrylate) (PMMA) gel swollen in CDCl3. Singular value decomposition (SVD) fitting analysis of all carbon-carbon bonds, 1DCC RDCs, in relation to the full configuration/conformational space (32 diastereoisomers) of artemisinin, unambiguously identified the correct configuration of artemisinin.

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Carbon-13 chemical shift tensor measurements for nitrogen-dense compounds.

This paper reports the principal values of the 13 C chemical shift tensors for five nitrogen-dense compounds (i.e., cytosine, uracil, imidazole, guanidine hydrochloride, and aminoguanidine hydrochloride). Although these are all fundamentally important compounds, the majority do not have 13 C chemical shift tensors reported in the literature. The chemical shift tensors are obtained from 1 H→13 C cross-polarization magic-angle spinning (CP/MAS) experiments that were conducted at a high field of 18.8T to suppress the effects of 14 N-13 C residual dipolar coupling. Quantum chemical calculations using density functional theory are used to obtain the 13 C magnetic shielding tensors for these compounds. The best agreement with experiment arises from calculations using the hybrid functional PBE0 or the double-hybrid functional PBE0-DH, along with the triple-zeta basis sets TZ2P or pc-3, respectively, and intermolecular effects modeled using large clusters of molecules with electrostatic embedding through the COSMO approach. These measurements are part of an ongoing effort to expand the catalog of accurate 13 C chemical shift tensor measurements, with the aim of creating a database that may be useful for benchmarking the accuracy of quantum chemical calculations, developing nuclear magnetic resonance (NMR) crystallography protocols, or aiding in applications involving machine learning or data mining. This work was conducted at the National High Magnetic Field Laboratory as part of a 2-week school for introducing undergraduate students to practical laboratory experience that will prepare them for scientific careers or postgraduate studies.

The effects of residual dipolar coupling on carnosine in proton muscle spectra.

Carnosine, an MR-visible dipeptide in human muscle, is well characterized by two peaks at ~8 and ~7 ppm from C2 and C4 imidazole protons. Like creatine and other metabolites, carnosine is subject to residual dipolar coupling in the anisotropic environment of muscle fibers, but the effects have not been studied extensively. Single-voxel TE 30-32 PRESS spectra from three different 3T studies were acquired from gastrocnemius medialis and soleus muscles in the human lower leg. In these studies, carnosine T2 values were measured, and spectra were obtained at three different foot angles. LCModel was used to fit the carnosine peaks with a basis set that was generated using shaped RF pulses and included a range of dipolar couplings affecting the C4 peak. A seven-parameter analytic expression was used to fit the CH2 doublets of creatine. It incorporated an optimized "effective TE" value to model the effect of shaped RF pulses. The fits confirm that the triplet C4 peak of carnosine is dipolar coupled to a pair of CH2 protons, with no need to include a contribution from a separate pool of freely rotating uncoupled carnosine. Moreover, the couplings experienced by carnosine C4 protons and creatine CH2 protons are strongly correlated (R2 = 0.88, P<0.001), exhibiting a similar 3cos2 θ - 1 dependence on the angle θ between fiber orientation and B0. T2 values for the singlet C2 peak of gastrocnemius carnosine are inversely proportional to the C4 dipolar coupling strength (R2 = 0.97, P < 0.001), which in turn is a function of foot orientation. This dependence indicates that careful positioning of the foot while acquiring lower leg muscle spectra is important to obtain reproducible carnosine concentrations. As proton magnetic resonance spectroscopy of carnosine is currently used to non-invasively estimate the muscle fiber typology, these results have important implications in sport science.

Exploring the impact of alignment media on RDC analysis of phosphorus-containing compounds: a molecular docking approach.

Residual dipolar couplings (RDCs) are employed in NMR analysis when conventional methods, such as J-couplings and nuclear Overhauser effects (NOEs) fail. Low-energy (optimized) conformers are often used as input structures in RDC analysis programs. However, these low-energy structures do not necessarily resemble conformations found in anisotropic environments due to interactions with the alignment medium, especially if the analyte molecules are flexible. Considering interactions with alignment media in RDC analysis, we developed and evaluated a molecular docking-based approach to generate more accurate conformer ensembles for compounds in the presence of the poly-γ-benzyl-L-glutamate alignment medium. We designed chiral phosphorus-containing compounds that enabled us to utilize 31P NMR parameters for the stereochemical analysis. Using P3D/PALES software to evaluate diastereomer discrimination, we found that our conformer ensembles outperform moderately the standard, low-energy conformers in RDC analysis. To further improve our results, we (i) averaged the experimental values of the molecular docking-based conformers by applying the Boltzmann distribution and (ii) optimized the structures through normal mode relaxation, thereby enhancing the Pearson correlation factor R and even diastereomer discrimination in some cases. Nevertheless, we presume that significant differences between J-couplings in isotropic and in anisotropic environments may preclude RDC measurements for flexible molecules. Therefore, generating conformer ensembles based on molecular docking enhances RDC analysis for mildly flexible systems while flexible molecules may require applying more advanced approaches, in particular approaches including dynamical effects.

Polyarylisocyanides Derived from an Alkyne-Pd(II) Catalyst as Robust Alignment Media with Excellent Enantiodiscimination.

The development of chiral alignment media for measuring anisotropic NMR parameters provides an opportunity to determine the absolute configuration of chiral molecules without the need for derivatization. However, chiral alignment media with a high and robust enantiodiscriminating property for a wide range of chiral molecules are still scarce. In this study, we synthesized cholesterol-end-functionalized helical polyisocyanides from a chiral monomer using a cholesterol-based alkyne-Pd(II) initiator. These stereoregular polyisocyanides form stable and weak anisotropic lyotropic liquid crystals (LLCs) in dichloromethane systems, exhibiting highly optical activities in both single left- and right-handed helices. The preparation process of the media was straightforward, and the aligning property of the LLCs could be controlled by adjusting the concentration and temperature. Using the chiral polyisocyanides, we extracted the residual dipolar coupling for an enantiomeric pair of isopinocampheol (IPC), as well as a number of pharmaceutical molecules, demonstrating excellent enantiodiscriminating properties for a broad range of chiral compounds.