Solid-state NMR Experiments Research Articles

Narrowing down the conformational space with solid-state NMR in crystal structure prediction of linezolid cocrystals

Many solids crystallize as microcrystalline powders, thus precluding the application of single crystal X-Ray diffraction in structural elucidation. In such cases, a joint use of high-resolution solid-state NMR and crystal structure prediction (CSP) calculations can be successful. However, for molecules showing significant conformational freedom, the CSP-NMR protocol can meet serious obstacles, including ambiguities in NMR signal assignment and too wide conformational search space to be covered by computational methods in reasonable time. Here, we demonstrate a possible way of avoiding these obstacles and making as much use of the two methods as possible in difficult circumstances. In a simple case, our experiments led to crystal structure elucidation of a cocrystal of linezolid (LIN), a wide-range antibiotic, with 2,3-dihydroxybenzoic acid, while a significantly more challenging case of a cocrystal of LIN with 2,4-dihydroxybenzoic acid led to the identification of the most probable conformations of LIN inside the crystal. Having four rotatable bonds, some of which can assume many discreet values, LIN molecule poses a challenge in establishing its conformation in a solid phase. In our work, a set of 27 conformations were used in CSP calculations to yield model crystal structures to be examined against experimental solid-state NMR data, leading to a reliable identification of the most probable molecular arrangements.

Structural Insights into the Interaction between Bacillus subtilis SepF Assembly and FtsZ by Solid-State NMR Spectroscopy.

In many species of Gram-positive bacteria, SepF participated in the membrane tethering of FtsZ Z-ring during bacteria division. However, atomic-level details of interaction between SepF and FtsZ in an assembled state are lacking. Here, by combining solid-state NMR (SSNMR) with biochemical analyses, the interaction of Bacillus subtilis SepF and the C-terminal domain (CTD) of FtsZ was investigated. We obtained near complete chemical shift assignments of SepF and determined the structural model of the SepF monomer. Interaction with FtsZ-CTD caused further packing of SepF rings, and SSNMR experiments revealed the affected residues locating at α1, α2, β3, and β4 of SepF. Solution NMR experiments of dimeric SepF constructed by point mutation strategy proved a prerequisite role of α-α interface formation in SepF for FtsZ binding. Overall, our results provide structural insights into the mechanisms of SepF-FtsZ interaction for better understanding the function of SepF in bacteria.

Revealing the roles of hydrocarbon pool mechanism in ethanol-to-hydrocarbons reaction

Ethanol-to-hydrocarbons (ETH) process, is investigated in this work due to the requirement of sustainable production route for light olefins such as ethene and propene. Majority of the mechanism insights of ETH process are principally proposed based on the methanol-to-hydrocarbons (MTH) process, which involves a homologous reaction system. The reaction intermediate species including cyclopentenyl cations and aromatics species, which are denoted as “hydrocarbon pool (HCP)” species in MTH process, were discovered and identified in the ETH process by in situ solid-state 13C NMR (ssNMR) experiments, advanced 2D 13C–13C INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment) ssNMR experiment, 12C/13C-C2H5OH isotope switching experiments complemented by gas chromatography-mass spectroscopy (GC–MS). Especially, in the ETH reaction, ethylcyclopentenyl cations and their deprotonated form were first captured and exhibited relatively higher activity in the formation of ethene and propene. Multi-routes with the participation of these active intermediates were proposed and evaluated by density functional theory calculation (DFT), demonstrating that they can play important roles in the formation of olefins. Ethene formation are mainly formed from ethanol dehydration, whereas propene can be produced via multi-routes with the participation of the captured HCP species. Moreover, the detailed reaction routes may be modulated by the temperature. This work provides direct evidences of the critical function of HCP species in the ETH process and reveals the mechanism of olefin formation.

In Situ 29Si solid-state NMR study of grafting of organoalkoxysilanes to mesoporous silica nanoparticles

Since the catalytic activity and the stability of silica-bound organometallic complexes are affected by their interactions with hydroxyl groups on the surface, isolated hydroxyls are often created prior to the introduction of catalytic species. Here, we investigate a method to remove the indigenous hydroxyls and create new isolated hydroxyls by grafting organo-trimethoxysilane (R-TMS) to generate a silicon T2 site, (≡SiO‒)2SiR(‒OH). We used in situ 29Si solid-state NMR experiments to monitor the evolution of Tn sites, (≡SiO‒)nSiR(‒OH)3-n (n = 1, 2, 3). The study indicates that i) the grafting proceeds in a consecutive manner as T1 → T2 → T3, and ii) the kinetics depend on the type of functional groups in the silane. However, the rates of T1 formation and T2 → T3 conversion are also controlled to a significant extent by the entropy loss associated to the initial silane binding and the spatial arrangement of surface hydroxyls, respectively. The grafting of R-TMS with a basic functional group leads to a lower concentration of T1 sites. The nucleophilicity of the functional group facilitates the grafting process by lowering the enthalpy barrier, while the T1 formation rate is more influenced by the entropy barrier than the T1 → T2 conversion rate. Thus, the basic functional group promotes the T1 → T2 conversion more than the T1 formation, resulting in a lower concentration of T1 sites.

Ultramicroporous Organophosphorus Polymers via Self-Accelerating P-C Coupling Reactions: Kinetic Effects on Crosslinking Environments and Porous Structures.

Porous organic polymers (POPs) have drawn significant attention in diverse applications. However, factors affecting the heterogeneous polymerization and porosity of POPs are still not well understood. Herein, we report a new strategy to construct porous organophosphorus polymers (POPPs) with high surface areas (1283 m2/g) and ultramicroporous structures (0.67 nm). The strategy harnesses an efficient transition-metal-catalyzed phosphorus-carbon (P-C) coupling reaction at the trigonal pyramidal P-center, which is distinct from the typical carbon-carbon coupling reaction utilized in the synthesis of POPs. As the first kinetic study on the coupling reaction of POPs, we uncovered a self-accelerating reaction characteristic, which is controlled by the choice of bases and catalysts. The self-accelerating characteristic of the P-C coupling reaction is beneficial for the high surface area and uniform ultramicroporosity of POPPs. The direct crosslinking of the P-centers allows 31P solid-state (ss)NMR experiments to unambiguously reveal the crosslinking environments of POPPs. Leveraging on the kinetic studies and 31P ssNMR studies, we were able to reveal the kinetic effects of the P-C coupling reaction on both the crosslinking environments and the porous structures of POPPs. Furthermore, our studies show that the CO2 uptake capacity of POPPs is highly dependent on their porous structures. Overall, our studies paves the way to design new POPs with better controlled chemical and ultramicroporous structures, which have potential applications for CO2 capture and separation.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Porous organic frameworks for preferable adsorption of trans-1,2-diols over cis-1,2-diols

• Porous organic frameworks for adsorption of vic- diols. • Preferable adsorption of trans-vic -diols over cis-vic -diols is possible. • Adsorption mechanism and selectivity depend on material structure. • MAS NMR to explore adsorption mechanisms. • Material application in a dynamic column setup. Novel separation processes are of utmost importance for a sustainable development of the chemical industry. In this work we show that polymer organic frameworks (POF) synthesized via condensation of melamine and a dialdehyde spacer exhibit superior selectivity towards adsorption of trans-vic -diols over cis - vic -diols. Mixtures of trans - and cis -1,2-cyclohexanediol as well as meso ‑ and ( R,R )-2,3-butanediol were applied for separation. Binding takes place due to cooperative hydrogen bonding of OH-groups of trans-diols to a nitrogen atom of the melamine ring and an NH-fragment of the aminal motif. The formation of the hydrogen bonds upon adsorption was supported by solid-state NMR experiments of samples after adsorption. Utilization of an aprotic solvent with low relative permittivity is of importance to achieve high adsorption capacities. The length of the dialdehyde spacer presents the major parameter governing the size of the micropores and thus the selectivity towards the trans -isomer. The best results were obtained for a POF prepared using 4,4′-biphenyldicarbaldehyde as spacer and bearing micropores with an average diameter of 0.67 nm. The average pore aperture is thus larger than the size of the tested diols, which are ca. 0.6 nm in length. Utilizing of isophtalaldehyde and phthalaldehyde as spacers gave rise to the sorption materials with average pore diameters of ca . 0.6 nm, comparable to the sizes of diol molecules. Adsorption of cis - and trans -diols onto the latter materials occurred owing to trapping in the pores, which resulted in strong van der Waals interactions leading to similar adsorption affinities of cis - and trans - isomers. The results of adsorption under batch and continuous conditions evidence the high potential of POF with 4,4′-biphenyldicarbaldehyde as spacer for adsorptive separation of trans - and cis -diols.

Ambiguous structure determination from powder data: four different structural models of 4,11-di-fluoro-quinacridone with similar X-ray powder patterns, fit to the PDF, SSNMR and DFT-D.

Four different structural models, which all fit the same X-ray powder pattern, were obtained in the structure determination of 4,11-di-fluoro-quinacridone (C20H10N2O2F2) from unindexed X-ray powder data by a global fit. The models differ in their lattice parameters, space groups, Z, Z', molecular packing and hydrogen bond patterns. The molecules form a criss-cross pattern in models A and B, a layer structure built from chains in model C and a criss-cross arrangement of dimers in model D. Nevertheless, all models give a good Rietveld fit to the experimental powder pattern with acceptable R-values. All molecular geometries are reliable, except for model D, which is slightly distorted. All structures are crystallochemically plausible, concerning density, hydrogen bonds, intermolecular distances etc. All models passed the checkCIF test without major problems; only in model A a missed symmetry was detected. All structures could have probably been published, although 3 of the 4 structures were wrong. The investigation, which of the four structures is actually the correct one, was challenging. Six methods were used: (1) Rietveld refinements, (2) fit of the crystal structures to the pair distribution function (PDF) including the refinement of lattice parameters and atomic coordinates, (3) evaluation of the colour, (4) lattice-energy minimizations with force fields, (5) lattice-energy minimizations by two dispersion-corrected density functional theory methods, and (6) multinuclear CPMAS solid-state NMR spectroscopy (1H, 13C, 19F) including the comparison of calculated and experimental chemical shifts. All in all, model B (perhaps with some disorder) can probably be considered to be the correct one. This work shows that a structure determination from limited-quality powder data may result in totally different structural models, which all may be correct or wrong, even if they are chemically sensible and give a good Rietveld refinement. Additionally, the work is an excellent example that the refinement of an organic crystal structure can be successfully performed by a fit to the PDF, and the combination of computed and experimental solid-state NMR chemical shifts can provide further information for the selection of the most reliable structure among several possibilities.

Practical guides for 1H detected solid-state NMR under fast MAS for small molecules

This primer article introduces a practical guide for setting up 1H detection solid-state NMR experiments under fast MAS conditions for small molecules. Sample independent adjustment/calibration including magic angle adjustment, shimming, radio-frequency field calibration, Hartmann-Hahn matching adjustments, and 1H heteronuclear decoupling are demonstrated. Experimental setups which depend on samples are also shown for 1H NMR, 1H-1H homonuclear correlation measurements, and 1H-X heteronuclear correlation/distance measurements. Information obtained from each experiment is also highlighted.

Elucidating structure and dynamics of crystalline α-zirconium phosphates intercalated with water and methanol by multinuclear solid-state MAS NMR: A comprehensive NMR approach.

Solid-state NMR experiments on 2 H, 31 P, 13 C, and 1 H nuclei, including 31 P T1 , 1 H T1 , and 1 H T1ρ measurements, as well as on the kinetics of proton-phosphorus cross-polarization have been performed to characterize the crystalline and amorphous α-zirconium phosphates, which were intercalated with D2 O and/or CD3 OD. The 13 C{1 H} CP MAS NMR experiment performed for compound 1-CD3 OD (Zr (HPO4 )2. 0.2CD3 OD) with carbon cross-polarization via protons of phosphate groups has provided a prove that the methanol was intercalated into the interlayer spaces of this compound. The variable-temperature 2 H solid-echo MAS NMR spectra of intercalated compounds demonstrated that the methanol molecules, in contrast to the mobile water, were immobile, keeping, however, free CD3 rotations around the C3 -axis. It has been demonstrated that the intercalated species, D2 O and CD3 OD, do not affect the high-frequency motions of the phosphate groups. By utilizing local structural models that satisfy the constraints of the experimental data, it has been suggested that the immobile methanol molecules are located in the cavity between two neighboring layers of the zirconium phosphates. Thus, the present work illustrates the reliable criteria in a comprehensive NMR approach to structural and dynamic studies of such systems.

Saponins Form Nonionic Lipid Nanodiscs for Protein Structural Studies by Nuclear Magnetic Resonance Spectroscopy.

Structural studies of membrane proteins in native-like environments require the development of diverse membrane mimetics. Currently there is a need for nanodiscs formed with nonionic belt molecules to avoid nonphysiological electrostatic interactions between the membrane system and protein of interest. Here, we describe the formation of lipid nanodiscs from the phospholipid DMPC and a class of nonionic glycoside natural products called saponins. The morphology, surface characteristics, and magnetic alignment properties of the saponin nanodiscs were characterized by light scattering and solid-state NMR experiments. We determined that preparing nanodiscs with high saponin/lipid ratios reduced their size, diminished their ability to spontaneously align in a magnetic field, and favored insertion of individual saponin molecules in the lipid bilayer surface. Further, purification of saponin nanodiscs allowed flipping of the orientation of aligned nanodiscs by 90°. Finally, we found that aligned saponin nanodiscs provide a sufficient alignment medium to allow the measurement of residual dipolar couplings (RDCs) in aqueous cytochrome c.

Fluidity is the way to life: lipid phase separation in bacterial membranes.

A hallmark of biological membranes is the dynamic localization of lipids and proteins. Lipids respond to temperature reduction below a critical point with phase separation, and poikilothermic animals and also bacteria adapt their lipid content to prevent gel phase formation in membranes. In a new study, Gohrbandt etal (2022) show that reduced membrane fluidity in bacterial cells causes reversible phase separation without membrane rupture invivo, highlighting the physical robustness of biological membranes.

In situ 13 C solid-state polarization transfer NMR to follow starch transformations in food.

Convenience food products tend to alter their quality and texture while stored. Texture-giving food components are often starch-rich ingredients, such as pasta or rice. Starch transforms depending on time, temperature and water content, which alters the properties of products. Monitoring these transformations, which are associated with a change in mobility of the starch chain segments, could optimize the quality of food products containing multiple ingredients. In order to do so, we applied a simple and efficient in situ 13 C solid-state magic angle spinning (MAS) NMR approach, based on two different polarization transfer schemes, cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT). The efficiency of the CP and INEPT transfer depends strongly on the mobility of chain segments-the time scale of reorientation of the CH-bond and the order parameter. Rigid crystalline or amorphous starch chains give rise to CP peaks, whereas mobile gelatinized starch chains appear as INEPT peaks. Comparing 13 C solid-state MAS NMR experiments based on CP and INEPT allows insight into the progress of gelatinization, and other starch transformations, by reporting on both rigid and mobile starch chains simultaneously with atomic resolution by the 13 C chemical shift. In conjunction with 1 H solid-state MAS NMR, complementary information about other food components present at low concentration, such as lipids and protein, can be obtained. We demonstrate our approach on starch-based products and commercial pasta as a function of temperature and storage.

Double echo symmetry-based REDOR and RESPDOR pulse sequences for proton detected measurements of heteronuclear dipolar coupling constants

1H{X} symmetry-based rotational echo double resonance pulse sequences (S-REDOR) and symmetry-based rotational echo saturation pulse double resonance (S-RESPDOR) solid-state NMR experiments have found widespread application for 1H detected measurements of difference NMR spectra, dipolar coupling constants, and internuclear distances under conditions of fast magic angle spinning (MAS). In these experiments the supercycled R412 (SR412) symmetry-based recoupling pulse sequence is typically applied to the 1H spins to reintroduce heteronuclear dipolar couplings. However, the timing of SR412 and other symmetry-based pulse sequences must be precisely synchronized with the rotation of the sample, otherwise, the evolution of 1H CSA and other interactions will not be properly refocused. For this reason, significant distortions are often observed in experimental dipolar dephasing difference curves obtained with S-REDOR or S-RESPDOR pulse sequences. Here we introduce a family of double echo (DE) S-REDOR/S-RESPDOR pulse sequences that function in an analogous manner to the recently introduced t1-noise eliminated (TONE) family of dipolar heteronuclear multiple quantum coherence (D-HMQC) pulse sequences. Through numerical simulations and experiments the DE S-REDOR/S-RESPDOR sequences are shown to provide dephasing difference curves similar to those obtained with S-REDOR/S-RESPDOR. However, the DE sequences are more robust to the deviations of the MAS frequency from the ideal value that occurs during typical solid-state NMR experiments. The DE sequences are shown to provide more reliable 1H detected dipolar dephasing difference curves for nuclei such as 15N (with isotopic labelling), 183W and 35Cl. The double echo sequences are therefore recommended to be used in place of conventional S-REDOR/S-RESPDOR sequences for measurement of weak dipolar coupling constants and long-range distances.

An ultra-dynamic anion-cluster-based organic framework

<h2>Summary</h2> Porous organic frameworks that can dynamically expand their voids to guest uptake remain formidable to design. The challenge comes from finding the balance between rigidity and flexibility. Rigidity is required to retain the framework, and the flexibility is needed for reversible expansion/contraction. Herein, we introduced bisulfate anion clusters as soft joints to construct a single-crystalline hydrogen-bonded cross-linked organic framework H_COF-6. Upon the uptake of guest molecules, the anion clusters of H_COF-6 were disrupted, allowing the crystal to expand rapidly (<40 min) to more than twice its original length. Removing these guests restored the anion clusters and the crystallinity of H_COF-6 quickly (<10 min). These guest-induced material size expansions and contractions are highly reversible, and the dynamic anion cluster dissociation-reassociation is confirmed by molecular dynamics simulations and solid-state NMR experiments. The guest-induced size change of H_COF-6 highlights a new approach to synthesize dynamic framework materials by introducing anion clusters.

Dynamics of quantum information scrambling under decoherence effects measured via active spin clusters

Developing quantum technologies requires the control and understanding of the non-equilibrium dynamics of quantum information in many-body systems. Local information propagates in the system by creating complex correlations known as information scrambling, as this process prevents extracting the information from local measurements. In this work, we develop a model adapted from solid-state NMR methods, to quantify the information scrambling. The scrambling is measured via time-reversal Loschmidt echoes (LE) and Multiple Quantum Coherences experiments that intrinsically contain imperfections. Considering these imperfections, we derive expressions for out-of-time-order correlators (OTOCs) to quantify the observable information scrambling based on measuring the number of active spins where the information was spread. Based on the OTOC expressions, decoherence effects arise naturally by the effects of the nonreverted terms in the LE experiment. Decoherence induces localization of the measurable degree of information scrambling. These effects define a localization cluster size for the observable number of active spins that determines a dynamical equilibrium. We contrast the model's predictions with quantum simulations performed with solid-state NMR experiments, that measure the information scrambling with time-reversal echoes with controlled imperfections. An excellent quantitative agreement is found with the dynamics of quantum information scrambling and its localization effects determined from the experimental data. The presented model and derived OTOCs set tools for quantifying the quantum information dynamics of large quantum systems (more than $10^{4}$ spins) consistent with experimental implementations that intrinsically contain imperfections.

Cholesterol-Mediated Clustering of the HIV Fusion Protein gp41 in Lipid Bilayers

The envelope glycoprotein (Env) of the human immunodeficient virus (HIV-1) is known to cluster on the viral membrane surface to attach to target cells and cause membrane fusion for HIV-1 infection. However, the molecular structural mechanisms that drive Env clustering remain opaque. Here, we use solid-state NMR spectroscopy and molecular dynamics (MD) simulations to investigate nanometer-scale clustering of the membrane-proximal external region (MPER) and transmembrane domain (TMD) of gp41, the fusion protein component of Env. Using 19F solid-state NMR experiments of mixed fluorinated peptides, we show that MPER-TMD trimers form clusters with interdigitated MPER helices in cholesterol-containing membranes. Inter-trimer 19F-19F cross peaks, which are indicative of spatial contacts within ∼2 nm, are observed in cholesterol-rich virus-mimetic membranes but are suppressed in cholesterol-free model membranes. Water-peptide and lipid-peptide cross peaks in 2D 1H-19F correlation spectra indicate that the MPER is well embedded in model phosphocholine membranes but is more exposed to the surface of the virus-mimetic membrane. These experimental results are reproduced in coarse-grained and atomistic molecular dynamics simulations, which suggest that the effects of cholesterol on gp41 clustering is likely via indirect modulation of the MPER orientation. Cholesterol binding to the helix-turn-helix region of the MPER-TMD causes a parallel orientation of the MPER with the membrane surface, thus allowing MPERs of neighboring trimers to interact with each other to cause clustering. These solid-state NMR data and molecular dynamics simulations suggest that MPER and cholesterol cooperatively govern the clustering of gp41 trimers during virus-cell membrane fusion.

A toolbox for improving the workflow of NMR crystallography

NMR crystallography is a powerful tool with applications in structural characterization and crystal structure verification, to name two. However, applying this tool presents several challenges, especially for industrial users, in terms of consistency, workflow, time consumption, and the requirement for a high level of understanding of experimental solid-state NMR and GIPAW-DFT calculations. Here, we have developed a series of fully parameterized scripts for use in Materials Studio and TopSpin, based on the .magres file format, with a focus on organic molecules (e.g. pharmaceuticals), improving efficiency, robustness, and workflow. We separate these tools into three major categories: performing the DFT calculations, extracting & visualizing the results, and crystallographic modelling. These scripts will rapidly submit fully parameterized CASTEP jobs, extract data from the calculations, assist in visualizing the results, and expedite the process of structural modelling. Accompanied with these tools is a description on their functionality, documentation on how to get started and use the scripts, and links to video tutorials for guiding new users. Through the use of these tools, we hope to facilitate NMR crystallography and to harmonize the process across users.

Sequence Determines the Switch in the Fibril Forming Regions in the Low-Complexity FUS Protein and Its Variants.

Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. Solid-state NMR experiments show that the 214-residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) have a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies.

Setting the magic angle using single crystal sapphire rotors

To date, the most common method for setting the magic angle in magic angle spinning (MAS) solid state NMR experiments has been to monitor the NMR signal from a quadrupolar nuclide (such as 79Br in KBr) under MAS, then swapping to a sample of interest. Here we introduce the use of single crystal sapphire rotors as a new method to set the magic angle while spinning a sample of interest. Using both simulations and experiment, we show that the 27Al satellite transitions for a single crystal sapphire rotor spinning about its crystalline c-axis manifest off magic angle as five resonances that coalesce into three peaks with maximum intensity at the magic angle. The difference in frequency between the satellite transitions can be used to measure the offset from the magic angle with high precision.