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.

Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins.

Large coupling networks in uniformly 13C,15N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer.

Band-selective universal 90° and 180° rotation pulses covering the aliphatic carbon chemical shift range for triple resonance experiments on 1.2 GHz spectrometers.

Biomolecular NMR spectroscopy requires large magnetic field strengths for high spectral resolution. Today's highest fields comprise proton Larmor frequencies of 1.2 GHz and even larger field strengths are to be expected in the future. In protein triple resonance experiments, various carbon bandwidths need to be excited by selective pulses including the large aliphatic chemical shift range. When the spectrometer field strength is increased, the length of these pulses has to be decreased by the same factor, resulting in higher rf-amplitudes being necessary in order to cover the required frequency region. Currently available band-selective pulses like Q3/Q5 excite a narrow bandwidth compared to the necessary rf-amplitude. Because the maximum rf-power allowed in probeheads is limited, none of the selective universal rotation pulses reported so far is able to cover the full [Formula: see text]C aliphatic region on 1.2 GHz spectrometers. In this work, we present band-selective 90° and 180° universal rotation pulses (SURBOP90 and SURBOP180) that have a higher ratio of selective bandwidth to maximum rf-amplitude than standard pulses. Simulations show that these pulses perform better than standard pulses, e. g. Q3/Q5, especially when rf-inhomogeneity is taken into account. The theoretical and experimental performance is demonstrated in offset profiles and by implementing the SURBOP pulses in an HNCACB experiment at 1.2 GHz.

Concurrent J-evolving refocusing pulses.

Conventional refocusing pulses are optimised for a single spin without considering any type of coupling. However, despite the fact that most couplings will result in undesired distortions, refocusing in delay-pulse-delay-type sequences with desired heteronuclear coherence transfer might be enhanced considerably by including coupling evolution into pulse design. We provide a proof of principle study for a Hydrogen-Carbon refocusing pulse sandwich with inherent J-evolution following the previously reported ICEBERG-principle with improved performance in terms of refocusing performance and/or overall effective coherence transfer time. Pulses are optimised using optimal control theory with a newly derived quality factor and z-controls as an efficient tool to speed up calculations. Pulses are characterised in theory and experiment and compared to conventional concurrent refocusing pulses, clearly showing an improvement for the J-evolving pulse sandwich. As a side-product, also efficient J-compensated resfocusing pulse sandwiches - termed BUBU pulses following the nomenclature of previous J-compensated BUBI and BEBEtr pulse sandwiches - have been optimised.

SORDOR pulses: expansion of the Böhlen-Bodenhausen scheme for low-power broadband magnetic resonance.

A novel type of efficient broadband pulse, called second-order phase dispersion by optimised rotation (SORDOR), has recently been introduced. In contrast to adiabatic excitation, SORDOR-90 pulses provide effective transverse 90∘ rotations throughout their bandwidth, with a quadratic offset dependence of the phase in the x,y plane. Together with phase-matched SORDOR-180 pulses, this enables the Böhlen-Bodenhausen broadband refocusing approach for linearly frequency-swept pulses to be extended to any type of 90∘/180∘ pulse-delay sequence. Example pulse shapes are characterised in theory and experiment, and an example application is given with a 19F-PROJECT experiment for measuring relaxation times with reduced distortions due to J-coupling evolution.

Selective 1 Hα NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome.

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two 1 Hα -detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes.

Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for 1H NMR assignment, by extending the high signal dispersion of 13C chemical shifts onto 1H resonances and by providing a high amount of through-bond 1H-1H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution 13C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the 13C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.

Selective excitation enables encoding and measurement of multiple diffusion parameters in a single experiment.

Band selectivity to address specific resonances in a spectrum enables one to encode individual settings for diffusion experiments. In a single experiment, this could include different gradient strengths (enabling coverage of a larger range of diffusion constants), different diffusion delays, or different gradient directions (enabling anisotropic diffusion measurement). In this report, a selective variant of the bipolar pulsed gradient eddy current delay (BPP-LED) experiment, enabling selective encoding of three resonances, was implemented. As proof of principle, the diffusion encoding gradient amplitude was assigned a range dependent on the selected signal, thereby allowing the extraction of the diffusion coefficient for water and a tripeptide (Met-Ala-Ser) with optimal settings in a single experiment.

Virtual decoupling to break the simplification versus resolution trade-off in nuclear magnetic resonance of complex metabolic mixtures.

The heteronuclear single quantum correlation (HSQC) experiment developed by Bodenhausen and Ruben (1980) in the early days of modern nuclear magnetic resonance (NMR) is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment, however, always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier and demonstrate its application towards metabolite identification in a complex mixture. Based on the measurement of clean in-phase and clean anti-phase (CLIP/CLAP) HSQC spectra (Enthart et al., 2008), we construct a virtually decoupled HSQC (vd-HSQC) spectrum that maintains the highest possible resolution in the proton dimension. Combining this vd-HSQC spectrum with a J-resolved spectrum (Pell and Keeler, 2007) provides useful information for the one-dimensional proton spectrum assignment and for the identification of metabolites in Dreissena polymorpha (Prud'homme et al., 2020).

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions.

Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic-resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water exchange, which disfavors classical amide 1H-detection, while 13C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of nonexchangeable 1Hα, so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this, we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the selective Hα,Cα-HSQC (SHACA-HSQC), using extensive hetero- and homonuclear decoupling applicable to aqueous samples (≥90% H2O) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to fivefold increased S/N compared to the standard 1H,13C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumor suppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any 1Hα-detected triple resonance experiment and may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC-Derived Tensorial Constraints*.

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility.

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19 F†NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

Urinary NMR Profiling in Pediatric Acute Kidney Injury-A Pilot Study.

Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations. In the study reported here, we examined the diagnostic accuracy of NMR urine metabolite patterns for the diagnosis of neonatal and pediatric AKI according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition. A cohort of 65 neonatal and pediatric patients (0-18 years) with established AKI of heterogeneous etiology was compared to both a group of apparently healthy children (n = 53) and a group of critically ill children without AKI (n = 31). Multivariate analysis identified a panel of four metabolites that allowed diagnosis of AKI with an area under the receiver operating characteristics curve (AUC-ROC) of 0.95 (95% confidence interval 0.86-1.00). Especially urinary citrate levels were significantly reduced whereas leucine and valine levels were elevated. Metabolomic differentiation of AKI causes appeared promising but these results need to be validated in larger studies. In conclusion, this study shows that NMR spectroscopy yields high diagnostic accuracy for AKI in pediatric patients.

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure 1 H-1 H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination.

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond 1 H-13 C RDCs alone often fall short. Long-range 1 H-1 H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign-sensitive way has been severely obstructed due to the overflow of 1 H-1 H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of 1 H-1 H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant-time ß-COSY experiment. The potential of 1 H-1 H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.

Configuration determination by residual dipolar couplings: accessing the full conformational space by molecular dynamics with tensorial constraints.

Residual dipolar couplings (RDCs) and other residual anisotropic NMR parameters provide valuable structural information of high quality and quantity, bringing detailed structural models of flexible molecules in solution in reach. The corresponding data interpretation so far is directly or indirectly based on the concept of a molecular alignment tensor, which, however, is ill-defined for flexible molecules. The concept is typically applied to a single or a small set of lowest energy structures, ignoring the effect of vibrational averaging. Here, we introduce an entirely different approach based on time averaged molecular dynamics with dipolar couplings as tensorial orientational restraints that can be used to solve structural problems in molecules of any size without the need of introducing an explicit molecular alignment tensor into the computation. RDC restraints are represented by their full 3D interaction tensor in the laboratory frame, for which pseudo forces are calculated using a secular dipolar Hamiltonian as the target. The resulting rotational averaging of each individual tensorial restraint leads to structural ensembles that best fulfil the experimental data. Using one-bond RDCs, the approach has been implemented in the force field procedures of the program COSMOS and extensively tested. A concise theoretical introduction, including the special treatment of force fields for stable and fast MD simulations, as well as applications regarding configurational analyses of small to medium-sized organic molecules with different degrees of flexibility, is given. The observed results are discussed in detail.

Molecular Dynamics with Orientational Tensorial Constraints: A New Approach to Probe the Torsional Angle Distributions of Small Rotationally Flexible Molecules.

The potential of residual dipolar couplings (RDCs) in conformational studies of small molecules is now widely recognized, but current theoretical approaches for their interpretation have several limitations and there is still the need for a general method to probe the torsional angle distributions applicable to any rotationally flexible molecule. Molecular dynamics simulations with RDC-based orientational tensorial constraints (MDOC), implemented in the software COSMOS, are presented here as a conceptually new strategy. For the cases of the fluorinated anti-inflammatory drug diflunisal and the disaccharide cellobiose, we demonstrate that MDOC simulations with one-bond RDCs as tensorial constraints unveil torsion distributions and allow the determination of relative configuration in the presence of rotational flexibility. The independence of the initial structure or any a priori assumption as well as the possibility to combine different experimental constraints represent features, which make the COSMOS software a promising tool for the investigation of torsional angle distributions of flexible molecules, regardless of their size and degree of freedom.

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage.

Intramembrane cleavage of the ß-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired γ-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ε-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.

Real-time pure shift measurements for uniformly isotope-labeled molecules using X-selective BIRD homonuclear decoupling.

We introduce a novel selective inversion element for chunked homonuclear decoupling that combines isotope selection via BIRD-filtering with band-selective inversion on the X-heteronucleus and allows efficient real-time decoupling of homonuclear and heteronuclear couplings. It is especially suitable for uniformly isotope-labeled compounds. We discuss in detail the inversion element based on band-selective refocusing on the X-nuclei (BASEREX), highlighting in particular the role of appropriate band-selective shaped refocusing pulses and the application of broadband X-pulses for an effective BIRDd element during homodecoupling. The approach is experimentally verified and studied in detail using uniformly 13C-labeled glucose and a uniformly 15N,13C-labeled amino acid mixture.

Efficient Extraction from Mice Feces for NMR Metabolomics Measurements with Special Emphasis on SCFAs.

Nuclear magnetic resonance (NMR) spectroscopy is one of the most promising methods for use in metabolomics studies as it is able to perform non targeted measurement of metabolites in a quantitative and non-destructive way. Sample preparation of liquid samples like urine or blood serum is comparatively easy in NMR metabolomics, because mainly buffer and chemical shift reference substance are added. For solid samples like feces suitable extraction protocols need to be defined as initial step, where the exact protocol depends on sample type and features. Focusing on short chain fatty acids (SCFAs) in mice feces, we describe here a set of extraction protocols developed with the aim to suppress changes in metabolite composition within 24 h after extraction. Feces are obtained from mice fed on either standard rodent diet or high fat diet. The protocols presented in this manuscript are straightforward for application, and successfully minimize residual bacterial and enzymatic activities. Additionally, they are able to minimize the lipid background originating from the high fat diet.

Increased H-Bond Stability Relates to Altered ε-Cleavage Efficiency and Aß Levels in the I45T Familial Alzheimer's Disease Mutant of APP.

Cleavage of the amyloid precursor protein's (APP) transmembrane domain (TMD) by γ-secretase is a crucial step in the aetiology of Alzheimer's Disease (AD). Mutations in the APP TMD alter cleavage and lead to familial forms of AD (FAD). The majority of FAD mutations shift the preference of initial cleavage from ε49 to ε48, thus raising the AD-related Aß42/Aß40 ratio. The I45T mutation is among the few FAD mutations that do not alter ε-site preference, while it dramatically reduces the efficiency of ε-cleavage. Here, we investigate the impact of the I45T mutation on the backbone dynamics of the substrate TMD. Amide exchange experiments and molecular dynamics simulations in solvent and a lipid bilayer reveal an increased stability of amide hydrogen bonds at the ζ- and γ-cleavage sites. Stiffening of the H-bond network is caused by an additional H-bond between the T45 side chain and the TMD backbone, which alters dynamics within the cleavage domain. In particular, the increased H-bond stability inhibits an upward movement of the ε-sites in the I45T mutant. Thus, an altered presentation of ε-sites to the active site of γ-secretase as a consequence of restricted local flexibility provides a rationale for reduced ε-cleavage efficiency of the I45T mutant.