Magic-angle Spinning Solid-state NMR Spectroscopy Research Articles

Resolving Atomic-Level Dynamics and Interactions of High-Molecular-Weight Hyaluronic Acid by Multidimensional Solid-State NMR.

High-molecular-weight (HMW) hyaluronic acid (HA) is a highly abundant natural polysaccharide and a fundamental component of the extracellular matrix (ECM). Its size and concentration regulate tissues' macro- and microenvironments, and its upregulation is a hallmark feature of certain tumors. Yet, the conformational dynamics of HMW-HA and how it engages with the components of the ECM microenvironment remain poorly understood at the molecular level. Probing the molecular structure and dynamics of HMW polysaccharides in a hydrated, physiological-like environment is crucial and also technically challenging. Here, we deploy advanced magic-angle spinning (MAS) solid-state NMR spectroscopy in combination with isotopic enrichment to enable an in-depth study of HMW-HA to address this challenge. This approach resolves multiple coexisting HA conformations and dynamics as a function of environmental conditions. By combining 13C-labeled HA with unlabeled ECM components, we detect by MAS NMR HA-specific changes in global and local conformational dynamics as a consequence of hydration and ECM interactions. These measurements reveal atom-specific variations in the dynamics and structure of the N-acetylglucosamine moiety of HA. We discuss possible implications for interactions that stabilize the structure of HMW-HA and facilitate its recognition by HA-binding proteins. The described methods apply similarly to the studies of the molecular structure and dynamics of HA in tumor contexts and in other biological tissues as well as HMW-HA hydrogels and nanoparticles used for biomedical and/or pharmaceutical applications.

Electrostatic Surface Potentials and Chalcogen-Bonding Motifs of Substituted 2,1,3-Benzoselenadiazoles Probed via 77Se Solid-State NMR Spectroscopy.

Chalcogen bonds (ChB) are moderately strong, directional, and specific non-covalent interactions that have garnered substantial interest over the last decades. Specifically, the presence of two σ-holes offers great potential for crystal engineering, catalysis, biochemistry, and molecular sensing. However, ChB applications are currently hampered by a lack of methods to characterize and control chalcogen bonds. Here, we report on the influence of various substituents (halogens, cyano, and methyl groups) on the observed self-complementary ChB networks of 2,1,3-benzoselenadiazoles. From molecular electrostatic potential calculations, we show that the electrostatic surface potentials (ESP) of the σ-holes on selenium are largely influenced by the electron-withdrawing character of these substituents. Structural analyses via X-ray diffraction reveal a variety of ChB geometries and binding modes that are rationalized via the computed ESP maps, although the structure of 5,6-dimethyl-2,1,3-benzoselenadiazole also demonstrates the influence of steric interactions. 77Se solid-state magic-angle spinning NMR spectroscopy, in particular the analysis of the selenium chemical shift tensors, is found to be an effective probe able to characterize both structural and electrostatic features of these self-complementary ChB systems. We find a positive correlation between the value of the ESP maxima at the σ-holes and the experimentally measured 77Se isotropic chemical shift, while the skew of the chemical shift tensor is established as a metric which is reflective of the ChB binding motif.

Optimal-Control-Based Cβ Chemical Shift Encoding for Efficient Signal Assignment of Solid Proteins.

Fast magic-angle spinning (MAS) solid-state NMR spectroscopy is a powerful tool for gaining structural and dynamic information on solid proteins. To access such information site-specifically, the signal assignment process is unavoidable. In the assignment process, Cα and Cβ chemical shifts are of paramount importance in identifying the type of amino acid residues. Conventionally, however, recording the Cβ chemical shift of solid proteins with relatively short transverse relaxation time is often limited by the long delay required for the magnetization transfer to Cβ spins and its evolution, that is, by the sensitivity drop. In this article, we propose a new method that encodes the Cβ chemical shifts onto the intensities of the scalar-coupled Cα signals by combining an optimal control-based spin manipulation pulse and a spin-state filter. This reduces the total required transverse evolution to less than half of that for the previously proposed method, opening up the concept of the Cβ-encoding nearest-neighbor NMR, for the first time, to solid proteins. Also, the total measurement time was shorter than that required for the explicit Cβ shift evolution. We demonstrate the sequential signal assignment for microcrystalline protein GB1, and then discuss the prospects for more challenging proteins.

Effect of borate, fluoride and strontium ions on biomimetic nucleation of calcium phosphate studied using solid-state nuclear magnetic resonance and X-ray diffraction

ObjectivesApatite minerals can have various anions and cations in their crystal structure in addition to phosphate ion (PO₄³⁻) and calcium ion (Ca2+). The aim of this study is to investigate effects of the borate, fluoride and strontium ions on biomimetic nucleation of calcium phosphate. MethodsNano-crystalline hydroxyapatite (H-Ap) was obtained from a supersaturated buffered solution containing 4.12 mM HPO42- and 5.88 mM Ca2+ (H-Ap). Four additives were used in solid solution methods: (i) 0.588 mM F- (F-Ap), (ii) 5.88 mM Sr2+ (Sr-Ap), (iii) 4.12 mM BO33- (BO3-Ap), and (iv) a surface pre-reacted glass ionomer (S-PRG) filler eluate that contained 0.17 mM Sr2+, 0.588 mM F-, 11.1 mM BO33-- (SPRG-Ap). Apatite crystallization was investigated using a solid-state magic-angle spinning NMR spectroscopy and X-ray diffraction (XRD) with the Rietveld analysis. ResultsA 2D 1H–31P heteronuclear-correlation NMR showed F- ion incorporation in the apatite structure of the F-Ap and SPRG-Ap. The peaks on the 31P axis of the F-Ap, Sr-Ap, and BO3-Ap were different from that of the H-Ap, and the full width at half maximum increased in the following order: H-Ap∼F-Ap∼BO3-Ap< SPRG-Ap< Sr-Ap, suggesting the incorporation of the F-, Sr2+ and BO33-. The incorporation of F and BO3 was further confirmed by 19F and 11B NMR. The XRD revealed that Sr2+ was preferentially incorporated into the CaII site. SignificanceThe F-, Sr2+ and BO33-ions might be involved in modifying the crystallization of apatite precipitation, producing a variety of apatite. S-PRG filler that release these ions may have an effect on remineralization, i.e., the reformation of apatite lost due to caries.

Serial Polarization Transfer by Combination of Cross-Relaxation and Rotational Resonance for Sensitivity-Enhanced Solid-State NMR.

Methods which induce site-specificity and sensitivity enhancement in solid-state magic-angle spinning NMR spectroscopy become more important for structural biology due to the increasing size of molecules under investigation. Recently, several strategies have been developed to increase site specificity and thus reduce signal overlap. Under dynamic nuclear polarization (DNP) for NMR signal enhancement, it is possible to use cross-relaxation transfer induced by select dynamic groups within the molecules which is exploited by SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions under DNP). Here, we present an approach where we additionally reintroduce the homonuclear dipolar coupling with rotational resonance (R2) during SCREAM-DNP to further boost the selectivity of the experiment. Detailed analysis of the polarization buildup dynamics of 13C-methyl polarization source and 13C-carbonyl target in 2-13C-ethyl 1-13C-acetate provides information about the sought-after and spurious transfer pathways. We show that dipolar-recoupled transfer rates greatly exceed the DNP buildup dynamics in our model system, indicating that significantly larger distances can be selectively and efficiently hyperpolarized.

Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR.

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43300-414), TDP-11 (TDP-43300-399), and TDP-10 (TDP-43314-414), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.

Initiated Chemical Vapor Deposited Anion-Conducting Solid-State Polymeric Electrolytes for All Solid-State Batteries: Impacts of Deposition Conditions and Polymer Composition on Performance Metrics

Achieving both high energy and power density, two traditionally opposed battery metrics, requires moving away from the 200+ years-old 2D layered battery configuration to a three-dimensional all solid-state battery (3D SSB) configuration. The enhanced power and energy benefits bestowed by a 3D SSB design have been demonstrated in the Li-ion 3D SSB literature.1 However, due to limitations in thermal and electronic conductivity of Li-Ion active materials, 3D Li-ion SSBs are limited to microscale dimensions. A potential means around this limitation is to switch to conductive metal electrodes such as Zn and Ag, for which 3D architectures have been demonstrated.2 In such a case, the remaining roadblock to a macroscale 3D SSB is the submicron-thick solid-state electrolyte, which requires a non-line-of-sight deposition method for incorporation into the complex 3D base electrode. Initiated chemical vapor deposition (iCVD) is a non–line-of-sight method that is ideal for generating conformal polymer coatings on complex 3D architectures. We focus on polymers amenable to iCVD protocols that can be modified post-deposition to introduce anion conduction pathways that can facilitate Ag and Zn redox. Submicron-thick coatings of poly-dimethylaminomethylstyrene (pDMAMS) that are pinhole-free and electronically insulating on both 2D planar and complex 3D electrode architectures are prepared via iCVD. The pDMAMS coatings are rendered anion-conducting through a vapor-phase methylation process and subsequent ion exchange to yield desirable anion species (e.g., OH–, Br–, or HCO3 –). X-ray photoelectron spectroscopy, ATR-IR spectroscopy, and solid-state magic-angle spinning NMR spectroscopy confirm the structure of the pDMAMS film before and after quaternization, while atomic force microscopy and cyclic voltammetry with redox probes confirm conformality and absence of pinholes in the submicron film. With the use of cyclic voltammetry and AC electrochemical impedance spectroscopy, the electronic and ionic conductivities of the polymer films are measured before and after quaternization. Molecular dynamics simulations of pDMAMS as a function of anion and H2O content in the film are used to generate computational results (e.g., glass-transition temperature Tg, anion conductivity) for comparison to experimental results.1. Long, J.W.; Dunn, B.; Rolison, D.R.; White, H.S. Chem. Rev. 2004, 104, 10, 4463–4492.2. Parker, J.F.; Chervin, C.N.; Nelson, E.S.; Rolison, D.R.; Long, J.W. Energy Environ Sci. 2014, 7, 1117-1124.

77Se and 125Te solid-state NMR and X-ray diffraction structural study of chalcogen-bonded 3,4-dicyano-1,2,5-chalcogenodiazole cocrystals.

Three novel chalcogen-bonded cocrystals featuring 3,4-dicyano-1,2,5-selenodiazole (C4N4Se) or 3,4-dicyano-1,2,5-tellurodiazole (C4N4Te) as chalcogen-bond donors and hydroquinone (C6H6O2), tetraphenylphosphonium chloride (C24H20P+·Cl-) or tetraethylphosphonium chloride (C8H20P+·Cl-) as chalcogen-bond acceptors have been prepared and characterized by single-crystal X-ray diffraction (XRD), powder X-ray diffraction and 77Se/125Te magic-angle spinning solid-state NMR spectroscopy. The single-crystal XRD results show that the chalcogenodiazole molecules interact with the electron donors through two σ-holes on each of the chalcogen atoms, which results in highly directional and moderately strong chalcogen bonds. Powder XRD confirms that the crystalline phases are preserved upon moderate grinding of the samples for solid-state NMR experiments. Measurement of 77Se and 125Te chemical shift tensors via magic-angle spinning solid-state NMR spectroscopy confirms the number of magnetically unique chalcogen sites in each asymmetric unit and reveals the impact of chalcogen-bond formation on the local electronic structure. These NMR data are further assessed in the context of analogous data for a wider range of crystalline chalcogen-bonded systems.

Assessment of halogen-bond induced cocrystallization of 1,3,5-trihalo-2,4,6-trifluorobenzenes with 2,3,5,6-Tetramethylpyrazine

Cocrystallization of each of 1,3,5-trichloro-2,4,6-trifluorobenzene, 1,3,5-tribromo-2,4,6-trifluorobenzene, and 1,3,5-triiodo-2,4,6-trifluorobenzene with 2,3,5,6-tetramethylpyrazine via selected mechanochemical and solution methods is assessed. In lieu of single crystals of suitable quality for structure determination via single-crystal X-ray diffraction, powder X-ray diffraction and 13C cross-polarization magic-angle spinning solid-state NMR spectroscopy are used to qualitatively assess halogen-bond induced cocrystal formation. The results suggest a rich polymorphic landscape worthy of further study.

NMR spectroscopy probes microstructure, dynamics and doping of metal halide perovskites.

Solid-state magic-angle spinning NMR spectroscopy is a powerful technique to probe atomic-level microstructure and structural dynamics in metal halide perovskites. It can be used to measure dopant incorporation, phase segregation, halide mixing, decomposition pathways, passivation mechanisms, short-range and long-range dynamics, and other local properties. This Review describes practical aspects of recording solid-state NMR data on halide perovskites and how these afford unique insights into new compositions, dopants and passivation agents. We discuss the applicability, feasibility and limitations of 1H, 13C, 15N, 14N, 133Cs, 87Rb, 39K, 207Pb, 119Sn, 113Cd, 209Bi, 115In, 19F and 2H NMR in typical experimental scenarios. We highlight the pivotal complementary role of solid-state mechanosynthesis, which enables highly sensitive NMR studies by providing large quantities of high-purity materials of arbitrary complexity and of chemical shifts calculated using density functional theory. We examine the broader impact of solid-state NMR on materials research and how its evolution over seven decades has benefitted structural studies of contemporary materials such as halide perovskites. Finally, we summarize some of the open questions in perovskite optoelectronics that could be addressed using solid-state NMR. We, thereby, hope to stimulate wider use of this technique in materials and optoelectronics research.

Initial Carbon-Carbon Bond Formation during the Early Stages of Methane Dehydroaromatization.

Methane dehydroaromatization (MDA) is among the most challenging processes in catalysis science owing to the inherent harsh reaction conditions and fast catalyst deactivation. To improve this process, understanding the mechanism of the initial C-C bond formation is essential. However, consensus about the actual reaction mechanism is still to be achieved. In this work, using advanced magic-angle spinning (MAS) solid-state NMR spectroscopy, we study in detail the early stages of the reaction over a well-dispersed Mo/H-ZSM-5 catalyst. Simultaneous detection of acetylene (i.e., presumably the direct C-C bond-forming product from methane), methylidene, allenes, acetal, and surface-formate species, along with the typical olefinic/aromatic species, allow us to conclude the existence of at least two independent C-H activation pathways. Moreover, this study emphasizes the significance of mobility-dependent host-guest chemistry between an inorganic zeolite and its trapped organic species during heterogeneous catalysis.

Two decades of progress in structural and dynamic studies of amyloids by solid-state NMR.

In this perspective article I briefly highlight the rapid progress made over the past two decades in atomic level structural and dynamic studies of amyloids, which are representative of non-crystalline biomacromolecular assemblies, by magic-angle spinning solid-state NMR spectroscopy. Given new and continuing developments in solid-state NMR instrumentation and methodology, ongoing research in this area promises to contribute to an improved understanding of amyloid structure, polymorphism, interactions, assembly mechanisms, and biological function and toxicity.

Interfacial Interactions Govern the Mechanisms of CO2 Absorption and Desorption on A2CO3-Promoted MgO (A = Na, K, Rb, and Cs) Absorbents

In this study, we investigated the detailed mechanisms of CO2 absorption and desorption on A2CO3-promoted MgO absorbents (A = Na, K, Rb, and Cs). We analyzed the materials formed at various stages of the absorbents during CO2 absorption and desorption by thermal decomposition-gas chromatography/mass spectroscopy, in situ IR spectroscopy, solid-state magic-angle spinning NMR spectroscopy on 13C, 23Na, 25Mg, and 39K nuclei, diffuse reflectance UV–vis spectroscopy, and in situ X-ray diffraction as well as the conventional thermogravimetric analysis, ex situ X-ray diffraction, and elemental distribution mapping by energy-dispersive X-ray analysis using scanning electron microscopy. The absorption of CO2 of the absorbents occurs in two steps. The first step is a fast process involving basic sites on the MgO surface formed by the interaction between A2CO3 and MgO. Because the basicity of these sites depends on the size of A ion, the kinetics and capacity of CO2 absorption and the desorption properties of this p...

Protein-solvent interfaces in human Y145Stop prion protein amyloid fibrils probed by paramagnetic solid-state NMR spectroscopy

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

Revealing the Local Proton Network through Three-Dimensional 13C/1H Double-Quantum/1H Single-Quantum and 1H Double-Quantum/13C/1H Single-Quantum Correlation Fast Magic-Angle Spinning Solid-State NMR Spectroscopy at Natural Abundance.

1H double quantum (DQ)/1H single quantum (SQ) correlation solid-state NMR spectroscopy is widely used to obtain internuclear 1H-1H proximities, especially at fast magic-angle spinning (MAS) rate (>60 kHz). However, to date, 1H signals are not well-resolved because of intense 1H-1H homonuclear dipolar interactions even at the attainable maximum MAS frequencies of ∼100 kHz and/or under 1H-1H homonuclear dipolar decoupling irradiations. Here we introduce novel three-dimensional (3D) experiments to resolve the 1H DQ/1H SQ correlation peaks using the additional 13C dimension. Although the low natural abundance of 13C (1.1%) significantly reduces the sensitivities, the 1H indirect measurements alleviate this issue and make this experiment possible even in naturally abundant samples. The two different implementations of 13C/1H DQ/1H SQ correlations and 1H DQ/13C/1H SQ correlations are discussed and demonstrated using l-histidine·HCl·H2O at natural abundance to reveal the local 1H-1H networks near each 13C. In addition, the complete 1H resonance assignments are achieved from a single 3D 13C/1H DQ/1H SQ experiment. We have also demonstrated the applicability of our proposed method on a biologically relevant molecule, capsaicin.

Proton-detected solid-state NMR spectroscopy of fully protonated proteins at slow to moderate magic-angle spinning frequencies

1H-detection offers a substitute to the sensitivity-starved experiments often used to characterize biomolecular samples using magic-angle spinning solid-state NMR spectroscopy (MAS-ssNMR). To mitigate the effects of the strong 1H–1H dipolar coupled network that would otherwise severely broaden resonances, high MAS frequencies (>40kHz) are often employed. Here, we have explored the alternative of stroboscopic 1H-detection at moderate MAS frequencies of 5–30kHz using windowed version of supercycled-phase-modulated Lee-Goldburg homonuclear decoupling. We show that improved resolution in the 1H dimension, comparable to that obtainable at high spinning frequencies of 40–60kHz without homonuclear decoupling, can be obtained in these experiments for fully protonated proteins. Along with detailed analysis of the performance of the method on the standard tri-peptide f-MLF, experiments on micro-crystalline GB1 and amyloid-β aggregates are used to demonstrate the applicability of these pulse-sequences to challenging biomolecular systems. With only two parameters to optimize, broadbanded performance of the homonuclear decoupling sequence, linear dependence of the chemical-shift scaling factor on resonance offset and a straightforward implementation under experimental conditions currently used for many biomolecular studies (viz. spinning frequencies and radio-frequency amplitudes), we expect these experiments to complement the current 13C-detection based methods in assignments and characterization through chemical-shift mapping.

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4. Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.

A method for dynamic nuclear polarization enhancement of membrane proteins.

Dynamic nuclear polarization (DNP) magic-angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin-labeled lipid molecules (SL-lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL4 . Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL4 reconstituted in liposomes containing SL-lipids reveal DNP enhancement values over two times larger for KL4 compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.

ChemInform Abstract: Recent Advances in Magic‐Angle Spinning Solid‐State NMR of Proteins

AbstractReview: 228 refs.

Membrane‐protein structure determination by solid‐state NMR spectroscopy of microcrystals: The autotransport domain of Yersinia YadA

Membrane proteins are largely underrepresented among available atomic‐resolution structures. The use of detergents in protein purification procedures hinders the formation of well‐ordered crystals for X‐ray crystallography and leads to slower molecular tumbling, impeding the application of solution‐state NMR. Solid‐state magic‐angle spinning NMR spectroscopy is an emerging method for membrane‐protein structural biology that can overcome these technical problems. Here we present the solid‐state NMR structure of the transmembrane domain of the Yersinia enterocolitica adhesin A (YadA). The sample was derived from crystallization trials that yielded only poorly diffracting microcrystals.We solved the structure using a single, uniformly 13C‐ and 15N‐labeled sample. In addition, solid‐state NMR allowed us to acquire information on the flexibility and mobility of parts of the structure, which, in combination with evolutionary conservation information, presents new insights into the autotransport mechanism of YadA.Funding: This work was supported by contract research ‘Forschungsprogramm Methoden für die Lebenswissenschaften’ of the Baden‐Württemberg Stiftung