A solid-state NMR study of cellulose degradation

A series of laboratory-aged transformer insulating papers were investigated using solid-state NMR spectroscopy. Carbon-13 CPMAS, and proton MAS experiments were carried out along with static proton relaxation (T₁, and T₁) and free induction decay (FID) measurements. Some proton CRAMPS and proton-carbon-13 correlation (WISE) experiments were also undertaken. A change in the proton T₁ and FID with ageing was detected. No detectable change was found in the proton T₁. Some amorphous cellulose was detected in the carbon-13 spectrum. There was, however, no evidence for a substantial change in the nature of the cellulose with ageing. The carbon-13 spectra from some aged samples showed signals not present in the spectrum from an unaged sample. This was taken to be evidence of chemical degradation. Proton MAS and the WISE exeriment gave some information about the nature of the water in the sample.     

NMR Spectroscopic Assignment of Backbone and Side‐Chain Protons in Fully Protonated Proteins: Microcrystals,Sedimented Assemblies,and Amyloid Fibrils

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid‐state NMR spectroscopy with 100–111 kHz magic‐angle spinning (MAS). The excellent resolution in the Cα‐Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence‐specific backbone and aliphatic side‐chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.     

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s⁻¹ was determined for residues protected from exchange. Transfer within the protein, as tracked in the ¹⁵N-¹H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s⁻¹ for several resolved residues, explaining the site specificity.     

Magic-angle-spinning-induced local ordering in polymer electrolytes and its effects on solid-state diffusion and relaxation NMR measurements

Magic-angle-spinning (MAS) enhances sensitivity and resolution in solid-state nuclear magnetic resonance (NMR) measurements. MAS is obtained by aerodynamic levitation and drive of a rotor, which results in large centrifugal forces that may affect the physical state of soft materials, such as polymers, and subsequent solid-state NMR measurements. Here, we investigate the effects of MAS on the solid-state NMR measurements of a polymer electrolyte for lithium-ion battery applications, poly(ethylene oxide) (PEO) doped with the lithium salt LiTFSI. We show that MAS induces local chain ordering, which manifests itself as characteristic lineshapes with doublet-like splittings in subsequent solid-state ¹ H, ⁷ Li, and ¹⁹ F static NMR spectra characterizing the PEO chains and solvated ions. MAS results in distributions of stresses and hence local chain orientations within the rotor, yielding distributions in the local magnetic susceptibility tensor that give rise to the observed NMR anisotropy and lineshapes. The effects of MAS were investigated on solid-state ⁷ Li and ¹⁹ F pulsed-field-gradient (PFG) diffusion and ⁷Li longitudinal relaxation NMR measurements. Activation energies for ion diffusion were affected modestly by MAS. ⁷Li longitudinal relaxation rates, which are sensitive to lithium-ion dynamics in the nanosecond regime, were essentially unchanged by MAS. We recommend that NMR researchers studying soft polymeric materials use only the spin rates necessary to achieve the desired enhancements in sensitivity and resolution, as well as acquire static NMR spectra after MAS experiments to reveal any signs of stress-induced local ordering.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, ¹H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi-acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D ¹H-detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de-excitation of specific resonances were achieved by high-fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U-¹³C,¹⁵N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel ¹H-detected multi-acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.     

Weak and Transient Protein Interactions Determined by Solid‐State NMR

Despite their roles in controlling many cellular processes, weak and transient interactions between large structured macromolecules and disordered protein segments cannot currently be characterized at atomic resolution by X‐ray crystallography or solution NMR. Solid‐state NMR does not suffer from the molecular size limitations affecting solution NMR, and it can be applied to molecules in different aggregation states, including non‐crystalline precipitates and sediments. A solid‐state NMR approach based on high magnetic fields, fast magic‐angle sample spinning, and deuteration provides chemical‐shift and relaxation mapping that enabled the characterization of the structure and dynamics of the transient association between two regions in an 80 kDa protein assembly. This led to direct verification of a mechanism of regulation of E. coli DNA metabolism.     

New and old NMR experiments for the resonance assignment of complex oligosaccharides--application to a cyclodextrin derivative

The complete assignment of the (1)H and (13)C sugar resonances in mono-3,6-anhydro-heptakis(2,3-O-methyl)-hexakis(6-O-methyl)-β-cyclodextrin, an asymmetrically functionalized β-cyclodextrin, was carried out by means of 2D NMR experiments. The TOCSY and the homonuclear multiple relay COSY spectra provided most of the (1)H assignments. The multiplicity edited HSQC and a set of F(1) selective HSQC-TOCSY and multiple relay HSQC-COSY spectra gave access to most of the (13)C chemical shifts. The latter were fully and accurately determined by means of a pair of complementary, highly folded HSQC-TOCSY spectra. The TOCSY-ROESY and ROESY-TOCSY spectra yielded the sequential assignment of the sugar units. A high resolution F(1) selective F(1) decoupled version of the TOCSY-ROESY experiment was recorded.     

Protein 3D structure determination by high-resolution solid-state NMR

Developments in NMR technology, sample preparation, pulse sequence methodology and structure calculation protocols have recently allowed one to progress towards structure determination at high-resolution of proteins by solid-state NMR spectroscopy. We here report solid-state NMR protocols based on magic-angle-spinning experiments, combined with modified structure calculation protocols, for structure determination of uniformly ¹³C, ¹⁵N isotopically labeled proteins. We demonstrate the use of these protocols to obtain high-resolution structures for the example of the microcrystalline Crh protein. The CHHC, DARR and PAR solid-state NMR experiments, as well as the calculation protocols using the program ARIA, are presented.     

1H NMR assignment and secondary structure of recombinant RGD-hirudin

The conformation of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was investigated by multi-dimensional NMR spectroscopy. The 1H NMR spectra of this protein are assigned in a sequential manner by using a combination of 2D NMR techniques to demonstrate through-bond and through-space (<5 A) connectivities. The secondary structure of recombinant RGD-hirudin was deduced from chemical shift indices, sequential NOEs and 3J(HNalpha) coupling constants. The results show that the recombinant RGD-hirudin has two anti-parallel beta-sheets and no alpha-helix, and also that the Arg-Gly-Asp (RGD) binding motif of this protein is located at the end of a long arm, which consists of two anti-parallel beta-strands (residues 26-31 and 36-41). As the strands are connected by a beta-turn, the recombinant RGD-hirudin acquires high flexibility and inhibits platelet aggregation more effectively.     

Stereospecific assignments of protein NMR resonances based on the tertiary structure and 2D/3D NOE data

In many cases of protein structure determination by NMR a high-quality structure is required. An important contribution to structural precision is stereospecific assignment of magnetically nonequivalent prochiral methylene and methyl groups, eliminating the need for introducing pseudoatoms and pseudoatom corrections in distance restraint lists. Here, we introduce the stereospecific assignment program that uses the resonance assignment, a preliminary 3D structure and 2D and/or 3D nuclear Overhauser effect spectroscopy peak lists for stereospecific assignment. For each prochiral group the algorithm automatically calculates a score for the two different stereospecific assignment possibilities, taking into account the presence and intensity of the nuclear Overhauser effect (NOE) peaks that are expected from the local environment of each prochiral group (i.e., the close neighbors). The performance of the algorithm has been tested and used on NMR data of alpha-helical and beta-sheet proteins using homology models and/or X-ray structures. The program produced no erroneous stereospecific assignments provided the NOEs were carefully picked and the 3D model was sufficiently accurate. The set of NOE distance restraints produced by nmr2st using the results of the SSA module was superior in generating good-quality ensembles of NMR structures (low deviations from upper limits in conjunction with low root-mean-square-deviation values) in the first round of structure calculations. The program uses a novel approach that employs the entire 3D structure of the protein to obtain stereospecific assignment; it can be used to speed up the NMR structure refinement and to increase the quality of the final NMR ensemble even when no scalar or residual dipolar coupling information is available.     

Hydrogen-bonded LC nanocomposites: characterisation of nanoparticle-LC interactions by solid-state NMR and FTIR spectroscopies

Although the molecular-level nanoparticle-liquid crystal (NP-LC) interactions are key for forming stable NP dispersions in LC, characterisation of these interactions is scarce in the literature. Recently, we have developed hydrogen-bonded LC nanocomposites based on mesogenic carboxylic acids and NPs functionalised with hydrogen-bond acceptor/donor groups. Here, we apply solid-state ¹H and ¹³C NMR and Fourier transform infrared spectroscopies to model systems consisting of mixtures of trans-4-n-butylcyclohexanecarboxylic acid (4-BCHA), 4-hexylbenzoic acid (6BA) and 4,4?-bipyridine. The binary mixture, 4-BCHA:6BA, was also studied. The results were compared to monolayer quantities of 6BA and 4-BCHA adsorbed on pyridine functionalised zirconia NPs (ZrO₂-n₃-pyridine) to understand the different effects of the functionalised NPs on the LC properties.     

Interdiffusion of PMMA and PVF2, studied by solid-state NMR

A previously published new solid-state nuclear magnetic resonance (NMR) method is applied to the interdiffusion of poly(methacrylate) (PMMA) and poly(vinylidene fluoride) (PVF₂) above their T_g. Via a variation of the cross-polarization technique magnetization is transferred from protons to fluorines. When this magnetization is made to disappear at the fluorine sites, only those protons that are distant from fluorines greater than the distance over which cross-polarization functions will retain their magnetization. In this way we detect the fraction of PMMA near (ca. 20 Å) PVF₂. Starting from sheets of PMMA and PVF₂, which are then heated at 190°C for a variable time, and applying the above technique, we can determine the fractions of PMMA and PVF₂ that have diffused within a distance of a few Å of each other. The intrinsic diffusion coefficients of PMMA and PVF₂ determined from such experiments compare well with literature data. Initial attempts to fit the experimental data suggest that the concentration dependence of the diffusion coefficients cannot be neglected. © 1994 John Wiley & Sons, Inc.     

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.