Multidimensional Solid-state Nuclear Magnetic Resonance Research Articles

Machine learning assisted interpretation of 2D solid-state nuclear magnetic resonance spectra

A machine learning methodology using deep neural network (DNN) for interpreting multidimensional solid-state nuclear magnetic resonance (SSNMR) of various synthetic and natural polymers is presented. The separated local field (SLF) SSNMR which correlates local well-defined heteronuclear dipolar with the tensor orientation of the chemical shift anisotropy (CSA) of spin in the solid state can provide valuable structure and molecular dynamics information of synthetic and biopolymers. Compared with the traditional linear least-square fitting, the proposed DNN-based methodology can efficiently and accurately determine the tensor orientation of CSA of both 13C and 15N in all four samples. The method achieves prediction precisions of the Euler angles with < ±5° and is characterized by low training costs and high efficiency (<1 s). The feasibility and robustness of the DNN-based analysis methodology are confirmed by comparison to reported-literature values. This strategy is expected to aid in the interpretation of complex multidimensional NMR spectra of complicated polymer system.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Solvent-derived defects suppress adsorption in MOF-74

Defects in metal-organic frameworks (MOFs) have great impact on their nano-scale structure and physiochemical properties. However, isolated defects are easily concealed when the frameworks are interrogated by typical characterization methods. In this work, we unveil the presence of solvent-derived formate defects in MOF-74, an important class of MOFs with open metal sites. With multi-dimensional solid-state nuclear magnetic resonance (NMR) investigations, we uncover the ligand substitution role of formate and its chemical origin from decomposed N,N-dimethylformamide (DMF) solvent. The placement and coordination structure of formate defects are determined by 13C NMR and density functional theory (DFT) calculations. The extra metal-oxygen bonds with formates partially eliminate open metal sites and lead to a quantitative decrease of N2 and CO2 adsorption with respect to the defect concentration. In-situ NMR analysis and molecular simulations of CO2 dynamics elaborate the adsorption mechanisms in defective MOF-74. Our study establishes comprehensive strategies to search, elucidate and manipulate defects in MOFs.

Electrochemical Complexation of Polyatomic Aluminum Cations in Quinone-Type Organic Battery Electrodes Revealed By Solid-State NMR

Rechargeable aluminum batteries represent a significant area of opportunity for beyond-lithium-ion energy storage due to the high theoretical capacity (2980 mA∙h∙g-1, 8046 mA∙h∙mL-1), large earth abundance (8.23 wt.%), low cost, and safety of aluminum metal. Due to the stable native oxide layer that forms on aluminum, very few electrolytes are able to reversibly plate and strip aluminum at room temperature. Lewis acidic chloroaluminate ionic liquid electrolytes have demonstrated success in this regard; however, any cathode materials must also be (electro)chemical stable in this electrolyte. Electrode materials based on organic molecules have garnered attention due to their tunable structures and properties, enabling optimization for specific energy storage systems. Furthermore, diverse functional groups enable a range of possible charge storage mechanisms, many of which have yet to be explored in these systems. Like aluminum, organic materials are highly abundant and can be derived from various sources without geopolitical constraints. However, few Al-organic battery studies exist, and none so far have performed mechanistic analyses based on experimental data to identify conclusively the electroactive ions and their binding sites.Here, we present a rigorous analysis of charge storage mechanisms in quinone-type organic molecules for the first time, established through multi-dimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy in concert with quantum chemical calculations and electrochemical measurements. Indanthrone quinone (INDQ) is investigated for the first time as a model system due to its two distinct quinone functionalities and low solubility in many solvents. Through-space 27Al{1H} and 13C{1H} dipolar-mediated ssNMR techniques are used to establish the sub-nanometer proximities between polyatomic aluminum cations and specific moieties in the organic frameworks. Furthermore, dipolar-mediated ssNMR measurements are sensitive to both molecular dynamics and internuclear distances, which enables us to selectively filter out signals from residual liquid electrolyte, eliminating the need for solvent rinsing that can perturb the sample. Al-INDQ cells achieve reversible capacities of up to 240 mA∙h∙g-1 and maintain capacities >100 mA∙h∙g-1 at rates up to 2400 mA∙g-1. The enolization reaction and charge-balancing AlCl2 + cation complexation was established via dipolar-mediated ssNMR, findings that are corroborated through density functional theory (DFT) calculations. Other quinone-based organic structures with modified structures and compositions were also investigated using similar methods and will briefly be compared to INDQ. In aggregate, the quinone-based model systems serve as a basis to better understand how different functional groups, heteroatoms, and organic structures affect the electrochemical properties of rechargeable Al-organic batteries. The results yield strategies towards creating ‘designer’ organic electrode materials for rechargeable aluminum batteries.

Molecular characterization of Dachengzi oil shale kerogen by multidimensional solid-state nuclear magnetic resonance spectroscopy

Solid-state nuclear magnetic resonance (SSNMR) techniques of 13C multiple cross polarization (multiCP), 13C multiCP with recoupled dipolar dephasing (multiCP/DD) and two-dimensional (2D) 1H–13C heteronuclear correlation (HETCOR) were used to characterize Dachengzi oil shale kerogen. Specific structural information was obtained from the quantitative multiCP and multiCP/DD spectra. The protonated aromatic signals distribute widely in the aromatic region and account for 24% of the total aromatic signals. The strong methylene peak in multiCP/DD spectrum indicates the long methylene chain structures. The 2D HETCOR spectra show that oxygen atoms play an important role in the connectivities between different groups. The strong peaks at the 1H/13C chemical shifts of 5/131 and 1.5/131 ppm indicate the high abundance of (–O–CH2)-aromatic and alkyl-substituted aromatic moieties. The connectivities were validated by calculating the chemical shifts of a series of relevant molecular models using quantum chemistry methods. The 13C signal assignment was completed and improved with the multidimensional SSNMR spectra. The results from SSNMR are in agreement with the fast pyrolysis study, and the appearance of (–O–CH2)-aromatic moieties indicates that the methylbenzene homologues in the pyrolysis products can originate from the cleavage of the C–O bonds in (–O–CH2)-aromatic moieties in addition to the β-scission reaction of linear alkylbenzene moieties.

SsNMRlib: a comprehensive library and tool box for acquisition of solid-state nuclear magnetic resonance experiments on Bruker spectrometers.

We introduce ssNMRlib, a comprehensive suite of pulse sequences and jython scripts for user-friendly solid-state nuclear magnetic resonance (NMR) data acquisition, parameter optimization and storage on Bruker spectrometers. ssNMRlib allows the straightforward setup of even highly complex multi-dimensional solid-state NMR experiments with a few clicks from an intuitive graphical interface directly from the Bruker Topspin acquisition software. ssNMRlib allows the setup of experiments in a magnetic-field-independent manner and thus facilitates the workflow in a multi-spectrometer setting with a centralized library. Safety checks furthermore assist the user in experiment setup. Currently hosting more than 140 1D to 4D experiments, primarily for biomolecular solid-state NMR, the library can be easily customized and new experiments are readily added as new templates. ssNMRlib is part of the previously introduced NMRlib library, which comprises many solution-NMR pulse sequences and macros.

Electrochemical Performance and Charge Storage Mechanism of Flavin-like Organic Electrodes for Rechargeable Aluminum Batteries

Rechargeable aluminum batteries represent a beacon of possibilities for sustainable electrochemical energy storage. Aluminum metal is energy dense, exhibiting high volumetric and gravimetric capacities of 8.05 A h cm-3 and 2.98 A h g-1, respectively. These capacities approximate to 75% of the volumetric and 400% of the gravimetric capacities of lithium metal. Additionally, aluminum is the most abundant metal in the Earth’s crust (8.23 wt. %), non-flammable, and benefits from mature mining, refinement, and recycling industries, thus making aluminum a low-cost alternative to lithium anodes in batteries. However, rechargeable aluminum batteries currently suffer from a scarcity of positive electrode materials (cathodes) that are both high performance and compatible with commonly used ionic liquid electrolytes. Organic molecules are derived from renewable and sustainable sources and can be leveraged as cathode materials; when paired with aluminum metal anodes, they result in batteries with earth abundant electrodes that are environmentally viable at scale.Here, we demonstrate a rechargeable aluminum battery using the organic molecule tetrathiobarbituric acid (TTBA) for the positive electrode and investigate its charge storage mechanism up from the molecular level through nuclear magnetic resonance (NMR) and electrochemical methods. For the electrolyte, a non-flammable, non-volatile ionic liquid was used, composed of AlCl3 and [EMIm]Cl (molar ratio: 1.5:1). The electrochemical performance of these cells was investigated through cyclic voltammetry and galvanostatic cycling techniques to determine reaction reversibility, cell capacity, and rate capability. Chiefly, the electrochemical results display >100 mA h g-1 for over 600 cycles at 100 mA g-1 and approximately 70 mA h g-1 at 500 mA g-1. The Al-TTBA batteries demonstrate long cycle lifetimes, yielding hundreds of cycles with significant capacity retention. This long cycle lifetime is attributed to TTBA’s low solubility in the electrolyte, a result of TTBA’s tetrameric structure.Molecular-level understanding of the charge storage mechanism was probed through spectroscopic and diffraction analyses, particularly multi-dimensional solid-state NMR measurements of TTBA electrodes conducted at different stages-of-charge. Solid-state 1H single-pulse and 13C{1H} cross-polarization (CP) magic-angle-spinning (MAS) NMR measurements yielded insights into local chemical, electronic, and structural changes that the TTBA undergoes upon ion complexation, while solid-state 27Al single-pulse NMR was used to probe aluminum coordination environments. Two-dimensional (2D) 27Al{1H} dipolar-mediated NMR correlation techniques, which probe sub-nanometer through-space proximities between 27Al and 1H moieties, were used to unambiguously establish the presence of aluminum bound to the electrode structure. X-ray diffraction (XRD) studies demonstrated changes in crystalline structure upon cycling.Overall, the results establish TTBA as a promising positive electrode material for rechargeable aluminum batteries, as well as yield molecular-level insights into its unique charge storage mechanism that are expected to aid the design of organic structures for multivalent-ion batteries.

Revealing Molecular Mechanisms in Hierarchical Nanoporous Carbon via Nuclear Magnetic Resonance

Author(s): Mao, H; Tang, J; Xu, J; Peng, Y; Chen, J; Wu, B; Jiang, Y; Hou, K; Chen, S; Wang, J; Lee, HR; Halat, DM; Zhang, B; Chen, W; Plantz, AZ; Lu, Z; Cui, Y; Reimer, JA | Abstract: Hierarchical nanoporous carbons combining pore sizes of different length scales are highly important for separation processes. However, critical questions remain regarding the hierarchical structure regulation and the molecular mechanisms of gaseous adsorbate uptake and interactions within the hierarchical nanoporous carbons. These materials present characterization challenges in that there are no experimental techniques that can elucidate the molecular mechanisms of the organic compounds and CO within the materials. We deployed multidimensional solid-state nuclear magnetic resonance (NMR) to generate maps of guest-framework interactions as a function of adsorbate concentrations and adsorption times. The NMR spectra provide insight toward the design of effective hierarchical pore structures. Our materials show a high volatile organic compounds/CO physisorption capacity, which reveals promising application to carbon-capture strategies to mitigate global warming. 2 2

Molecular-Scale Understanding of Charge Storage Mechanisms in Positive Electrode Materials for Rechargeable Aluminum Metal Batteries

Rechargeable aluminum metal batteries are an emerging energy storage technology with great promise: aluminum has among the highest capacities of common metal electrodes and is low cost, earth abundant, environmentally friendly, and inherently safe. Despite these opportunities, their technological development has been hindered due to fundamental challenges associated with aluminum electrochemistry. Few electrolytes enable the reversible electrodeposition of aluminum metal at room temperature, while few positive electrode materials have been demonstrated that exhibit high energy density and cycle life in those electrolytes. Here, recent progress will be discussed in the development and characterization of positive electrode materials for rechargeable aluminum metal batteries, including graphites, organic materials, sulfur, and crystalline transition metal compounds. Molecular-scale understanding of their charge storage mechanisms will be elucidated, revealed by a combination of electrochemical, spectroscopic, diffraction, imaging, and theoretical methods, such as multi-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, in operando X-ray diffraction (XRD), electron microscopy, and density functional theory (DFT). The diversity of electrochemical charge storage mechanisms possible for aluminum battery chemistries will be highlighted. Overall, the results are aimed at developing next-generation rechargeable aluminum metal batteries for diverse energy storage applications.

Transferring principles of solid-state and Laplace NMR to the field of in vivo brain MRI.

Magnetic resonance imaging (MRI) is the primary method for noninvasive investigations of the human brain in health, disease, and development but yields data that are difficult to interpret whenever the millimeter-scale voxels contain multiple microscopic tissue environments with different chemical and structural properties. We propose a novel MRI framework to quantify the microscopic heterogeneity of the living human brain as spatially resolved five-dimensional relaxation-diffusion distributions by augmenting a conventional diffusion-weighted imaging sequence with signal encoding principles from multidimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, relaxation-diffusion correlation methods from Laplace NMR of porous media, and Monte Carlo data inversion. The high dimensionality of the distribution space allows resolution of multiple microscopic environments within each heterogeneous voxel as well as their individual characterization with novel statistical measures that combine the chemical sensitivity of the relaxation rates with the link between microstructure and the anisotropic diffusivity of tissue water. The proposed framework is demonstrated on a healthy volunteer using both exhaustive and clinically viable acquisition protocols.

Unlocking the architecture of native plant cell walls via solid-state nuclear magnetic resonance.

Understanding the biosynthesis and architecture of the plant cell wall is key to predictably engineering plants as a sustainable bioenergy source. Multi-dimensional solid-state nuclear magnetic resonance (ssNMR) is a promising technique to investigate the three-dimensional networks formed between cell wall components in their native state, and develop models of cell wall architecture. To do this, it is necessary to produce plant material that is highly enriched in the carbon-13 isotope (13C), since carbon-12 (12C) is inactive in NMR. Here, we present a cost-effective way to generate 13C-enriched mature plant tissue and to determine the degree of 13C incorporation. We describe a series of multi-dimensional ssNMR experiments that have been used in recent studies of cell wall architecture, and provide an introduction to interpreting the resulting ssNMR spectra.

Resolution enhancement and proton proximity probed by 3D TQ/DQ/SQ proton NMR spectroscopy under ultrafast magic-angle-spinning beyond 70 kHz.

Proton nuclear magnetic resonance (NMR) in solid state has gained significant attention in recent years due to the remarkable resolution and sensitivity enhancement afforded by ultrafast magic-angle-spinning (MAS). In spite of the substantial suppression of 1H-1H dipolar couplings, the proton spectral resolution is still poor compared to that of 13C or 15N NMR, rendering it challenging for the structural and conformational analysis of complex chemicals or biological solids. Herein, by utilizing the benefits of double-quantum (DQ) and triple-quantum (TQ) coherences, we propose a 3D single-channel pulse sequence that correlates proton triple-quantum/double-quantum/single-quantum (TQ/DQ/SQ) chemical shifts. In addition to the two-spin proximity information, this 3D TQ/DQ/SQ pulse sequence enables more reliable extraction of three-spin proximity information compared to the regular 2D TQ/SQ correlation experiment, which could aid in revealing the proton network in solids. Furthermore, the TQ/DQ slice taken at a specific SQ chemical shift only reveals the local correlations to the corresponding SQ chemical shift, and thus it enables accurate assignments of the proton peaks along the TQ and DQ dimensions and simplifies the interpretation of proton spectra especially for dense proton networks. The high performance of this 3D pulse sequence is well demonstrated on small compounds, L-alanine and a tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (MLF). We expect that this new methodology can inspire the development of multidimensional solid-state NMR pulse sequences using the merits of TQ and DQ coherences and enable high-throughput investigations of complex solids using abundant protons.

Optimization of band-selective homonuclear dipolar recoupling in solid-state NMR by a numerical phase search.

Spin polarization transfers among aliphatic 13C nuclei, especially 13Cα-13Cβ transfers, permit correlations of their nuclear magnetic resonance (NMR) frequencies that are essential for signal assignments in multidimensional solid-state NMR of proteins. We derive and demonstrate a new radio-frequency (RF) excitation sequence for homonuclear dipolar recoupling that enhances spin polarization transfers among aliphatic 13C nuclei at moderate magic-angle spinning (MAS) frequencies. The phase-optimized recoupling sequence with five π pulses per MAS rotation period (denoted as PR5) is derived initially from systematic numerical simulations in which only the RF phases are varied. Subsequent theoretical analysis by average Hamiltonian theory explains the favorable properties of numerically optimized phase schemes. The high efficiency of spin polarization transfers in simulations is preserved in experiments, in part because the RF field amplitude in PR5 is only 2.5 times the MAS frequency so that relatively low 1H decoupling powers are required. Experiments on a microcrystalline sample of the β1 immunoglobulin binding domain of protein G demonstrate an average enhancement factor of 1.6 for 13Cα → 13Cβ polarization transfers, compared to the standard 13C-13C spin-diffusion method, implying a two-fold time saving in relevant 2D and 3D experiments.

Duet of Acetate and Water at the Defects of Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) are porous crystalline materials with promising applications in molecular adsorption, separation, and catalysis. It has been discovered recently that structural defects introduced unintentionally or by design could have a significant impact on their properties. However, the exact chemical composition and structural evolution under different conditions at the defects are still under debate. In this study, we performed multidimensional solid-state nuclear magnetic resonance (SSNMR) coupled with computer simulations to elucidate an important scenario of MOF defects, uncovering the dynamic interplay between residual acetate and water. Acetate, as a defect modulator, and water, as a byproduct, are prevalent defect-associated species, which are among the key factors determining the reactivity and stability of defects. We discovered that acetate molecules coordinate to a single metal site monodentately and pair with water at the neighboring position. The acetates are highly flexible, which undergo fast libration as well as a slow kinetic exchange with water through dynamic hydrogen bonds. The dynamic processes under variable temperatures and different hydration levels have been quantitatively analyzed across a broad time scale from microseconds to seconds. The integration of SSNMR and computer simulations allows a precision probe into defective MOF structures with intrinsic dynamics and disorder.

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Solid-State NMR Characterization of Mixed Phosphonic Acid Ligand Binding and Organization on Silica Nanoparticles.

As ligand functionalization of nanomaterials becomes more complex, methods to characterize the organization of multiple ligands on surfaces is required. In an effort to further the understanding of ligand-surface interactions, a combination of multinuclear ((1)H, (29)Si, (31)P) and multidimensional solid-state nuclear magnetic resonance (NMR) techniques was utilized to characterize the phosphonic acid functionalization of fumed silica nanoparticles using methylphosphonic acid (MPA) and phenylphosphonic acid (PPA). (1)H → (29)Si cross-polarization (CP)-magic angle spinning (MAS) solid-state NMR was used to selectively detect silicon atoms near hydrogen atoms (primarily surface species); these results indicate that geminal silanols are preferentially depleted during the functionalization with phosphonic acids. (1)H → (31)P CP-MAS solid-state NMR measurements on the functionalized silica nanoparticles show three distinct resonances shifted upfield (lower ppm) and broadened compared to the resonances of the crystalline ligands. Quantitative (31)P MAS solid-state NMR measurements indicate that ligands favor a monodentate binding mode. When fumed silica nanoparticles were functionalized with an equal molar ratio of MPA and PPA, the MPA bound the nanoparticle surface preferentially. Cross-peaks apparent in the 2D (1)H exchange spectroscopy (EXSY) NMR measurements of the multiligand sample at short mixing times indicate that the MPA and PPA are spatially close (≤5 Å) on the surface of the nanostructure. Furthermore, (1)H-(1)H double quantum-single quantum (DQ-SQ) back-to-back (BABA) 2D NMR spectra further confirmed that MPA and PPA are strongly dipolar coupled with observation of DQ intermolecular contacts between the ligands. DQ experimental buildup curves and simulations indicate that the average distance between MPA and PPA is no further than 4.2 ± 0.2 Å.

Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary Cell Walls: Evidence from Solid-State Nuclear Magnetic Resonance

The structural role of pectins in plant primary cell walls is not yet well understood because of the complex and disordered nature of the cell wall polymers. We recently introduced multidimensional solid-state nuclear magnetic resonance spectroscopy to characterize the spatial proximities of wall polysaccharides. The data showed extensive cross peaks between pectins and cellulose in the primary wall of Arabidopsis (Arabidopsis thaliana), indicating subnanometer contacts between the two polysaccharides. This result was unexpected because stable pectin-cellulose interactions are not predicted by in vitro binding assays and prevailing cell wall models. To investigate whether the spatial contacts that give rise to the cross peaks are artifacts of sample preparation, we now compare never-dried Arabidopsis primary walls with dehydrated and rehydrated samples. One-dimensional (13)C spectra, two-dimensional (13)C-(13)C correlation spectra, water-polysaccharide correlation spectra, and dynamics data all indicate that the structure, mobility, and intermolecular contacts of the polysaccharides are indistinguishable between never-dried and rehydrated walls. Moreover, a partially depectinated cell wall in which 40% of homogalacturonan is extracted retains cellulose-pectin cross peaks, indicating that the cellulose-pectin contacts are not due to molecular crowding. The cross peaks are observed both at -20 °C and at ambient temperature, thus ruling out freezing as a cause of spatial contacts. These results indicate that rhamnogalacturonan I and a portion of homogalacturonan have significant interactions with cellulose microfibrils in the native primary wall. This pectin-cellulose association may be formed during wall biosynthesis and may involve pectin entrapment in or between cellulose microfibrils, which cannot be mimicked by in vitro binding assays.

Paramagnetic Ions Enable Tuning of Nuclear Relaxation Rates and Provide Long-Range Structural Restraints in Solid-State NMR of Proteins

Magic-angle-spinning solid-state nuclear magnetic resonance (SSNMR) studies of natively diamagnetic uniformly (13)C,(15)N-enriched proteins, intentionally modified with side chains containing paramagnetic ions, are presented, with the aim of using the concomitant nuclear paramagnetic relaxation enhancements (PREs) as a source of long-range structural information. The paramagnetic ions are incorporated at selected sites in the protein as EDTA-metal complexes by introducing a solvent-exposed cysteine residue using site-directed mutagenesis, followed by modification with a thiol-specific reagent, N-[S-(2-pyridylthio)cysteaminyl]EDTA-metal. Here, this approach is demonstrated for the K28C and T53C mutants of B1 immunoglobulin-binding domain of protein G (GB1), modified with EDTA-Mn(2+) and EDTA-Cu(2+) side chains. It is shown that incorporation of paramagnetic moieties, exhibiting different relaxation times and spin quantum numbers, facilitates the convenient modulation of longitudinal (R(1)) and transverse (R(2), R(1rho)) relaxation rates of the protein (1)H, (13)C, and (15)N nuclei. Specifically, the EDTA-Mn(2+) side chain generates large distance-dependent transverse relaxation enhancements, analogous to those observed previously in the presence of nitroxide spin labels, while this phenomenon is significantly attenuated for the Cu(2+) center. Both Mn(2+) and Cu(2+) ions cause considerable longitudinal nuclear PREs. The combination of negligible transverse and substantial longitudinal relaxation enhancements obtained with the EDTA-Cu(2+) side chain is especially advantageous, because it enables structural restraints for most sites in the protein to be readily accessed via quantitative, site-resolved measurements of nuclear R(1) rate constants by multidimensional SSNMR methods. This is demonstrated here for backbone amide (15)N nuclei, using methods based on 2D (15)N-(13)C chemical shift correlation spectroscopy. The measured longitudinal PREs are found to be highly correlated with the proximity of the Cu(2+) ion to (15)N spins, with significant effects observed for nuclei up to approximately 20 A away, thereby providing important information about protein structure on length scales that are inaccessible to traditional SSNMR techniques.

Multidimensional solid-state nuclear magnetic resonance for determining the dihedral angle from the correlation of C13–H1 and C13–C13 dipolar interactions under magic-angle spinning conditions

Multidimensional solid-state nuclear magnetic resonance (NMR) under magic-angle spinning (MAS) conditions has been developed to determine the dihedral angle for a Hα1–Cα13–Cβ13–Hβ1 moiety in powdered states. The pulse sequence for this experiment includes C113H dipolar evolution periods for Cα and Cβ, which are correlated through a coherent Cα1313Cβ dipolar mixing period. Theoretical analysis based on the symmetry of the spin system indicates that the dipolar correlation spectrum only due to the CαHα and CβHβ dipolar couplings is strongly dependent on the dihedral angle χ about the CαCβ bond axis, but two χ angles give the same spectrum in the χ range from 0° to about 140°, where χ=0° corresponds to the cis conformation. Inclusion of the CαCβ dipolar coupling together with the weak CαHβ and CβHα dipolar couplings, however, breaks the symmetry of the system with respect to χ in the range from 0° to 180°. These properties are confirmed by the spectra calculated for the pulse sequence as a function of χ and the root-mean-square deviation between them. The bond lengths, bond angles, and dihedral angle also alter the dipolar correlation spectrum differently. This enables us the experimental determination of all the structural parameters, which improves the accuracy of the dihedral angle determination. The high resolution due to C13 isotropic chemical shifts under MAS conditions in this multidimensional NMR permits its application to molecules having a number of C13-labeled sites. Experimental results are presented for powdered L-valine uniformly labeled with C13 and N15 nuclei. Effects of the structural parameters and noise on the dihedral angle determination are evaluated numerically. The accuracies of the determined structural parameters are discussed.

Different dynamic filters constructed from multidimensional NMR experiments

The design of specific dynamic filters on the basis of different multi-time correlation functions as measured by multidimensional solid-state nuclear magnetic resonance spectroscopy is discussed. Supplementary to previous applications of this type of experiment, not only slow dynamics but also fast relaxation processes are selected and their dynamic properties are analysed. The experiment is applied to the non-exponential structural relaxation of poly(vinyl acetate). Dynamic heterogeneities are shown to dominate the relaxation. Equilibration of both fast and slow subensembles is observed on the same time scale.