We report dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their life cycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic-level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking. We observed up to 64-fold signal enhancements by DNP, which permitted in-depth analysis of these assemblies. For the HIV-1 CA assemblies, a remarkably high spectral resolution in the 3D and 2D heteronuclear data sets permitted the assignment of a significant fraction of backbone and side-chain resonances. Using an integrated DNP MAS NMR and molecular dynamics (MD) simulation approach, the conformational space sampled by the assembled capsid at cryogenic temperatures was mapped. Qualitatively, a remarkable agreement was observed for the experimental 13C/15N chemical shift distributions and those calculated from substructures along the MD trajectory. Residues that are mobile at physiological temperatures are frozen out in multiple conformers at cryogenic conditions, resulting in broad experimental and calculated chemical shift distributions. Overall, our results suggest that DNP MAS NMR measurements in combination with MD simulations facilitate a thorough understanding of the dynamic signatures of viral capsids.

The mature conical HIV-1 capsid, comprising approximately 1300 copies of the CA protein, houses the viral genome and plays multiple roles during viral infection. Although the capsid has been extensively studied by various methodologies, an atomic resolution structure is still lacking. We employed magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the atomic-resolution structure of CA tubular assemblies. A large number of intramolecular and intermolecular 13C-13C and 13C-15N distance restraints, up to 7 Å, were obtained from a suite of 2D and 3D homo- and heteronuclear MAS NMR experiments using sets of sparsely and differentially isotopically labeled CA tubes, from which the 3D structure of CA monomer was determined. The CA protein comprises two independently folded domains, and the relative domain positions were not well defined in our structure, due to the dearth of long-range restraints. Therefore, we further explored 19F as a novel NMR structural probe. Tryptophan residues with fluorine substitutions in the 5 position of the indole ring were used as the reporters. Spin diffusion and radio frequency driven recoupling experiments were performed at MAS frequencies of 35 kHz and 40-60 kHz, respectively. Fast MAS frequencies of 40-60 kHz are essential for consistently establishing 19F-19F correlations, yielding interatomic distances of the order of 20 Å. By combining the 19F-19F distances with the conventional 13C-13C and 13C-15N distances, we are moving towards the atomic-resolution structure of the HIV-1 capsid assemblies.

We report remarkably high, up to 100-fold, signal enhancements in 19F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 capsid protein (CA) assemblies. These enhancements correspond to absolute sensitivity ratios of 12-29 and are of similar magnitude to those seen for 1H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D 19F-13C HETCOR spectra, which contain long-range intra- and intermolecular correlations. Such correlations provide unique distance restraints, inaccessible in conventional experiments without DNP, for protein structure determination. Furthermore, systematic quantification of the DNP enhancements as a function of biradical concentration, MAS frequency, temperature, and microwave power is reported. Our work establishes the power of DNP-enhanced 19F MAS NMR spectroscopy for structural characterization of HIV-1 CA assemblies, and this approach is anticipated to be applicable to a wide range of large biomolecular systems.

Abstract19F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS) 19F solid‐state NMR spectroscopy in assemblies of HIV‐1 capsid protein. Tryptophan residues with fluorine substitution at the 5‐position of the indole ring were used as the reporters. The 19F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin‐diffusion and radio‐frequency‐driven‐recoupling experiments were performed at MAS frequencies of 35 kHz and 40–60 kHz, respectively. Fast MAS frequencies of 40–60 kHz are essential for consistently establishing 19F–19F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS 19F NMR spectroscopy for structural analysis in large biological assemblies.

The 19F chemical shift is a sensitive NMR probe of structure and electronic environment in organic and biological molecules. In this report, we examine chemical shift parameters of 4F-, 5F-, 6F-, and 7F-substituted crystalline tryptophan by magic angle spinning (MAS) solid-state NMR spectroscopy and density functional theory. Significant narrowing of the 19F lines was observed under fast MAS conditions, at spinning frequencies above 50 kHz. The parameters characterizing the 19F chemical shift tensor are sensitive to the position of the fluorine in the aromatic ring and, to a lesser extent, the chirality of the molecule. Accurate calculations of 19F magnetic shielding tensors require the PBE0 functional with a 50% admixture of a Hartree-Fock exchange term, as well as taking account of the local crystal symmetry. The methodology developed will be beneficial for 19F-based MAS NMR structural analysis of proteins and protein assemblies.

Planar microcoils with diameter ranging from 20 to 1000 μm I.D. (130-1130 μm O.D.) are evaluated for their applications in NMR spectroscopy. The coils are first overfilled with a standard sucrose solution and compared against each other. Coils with smaller I.D. (≤100 μm) perform extremely well. One hypothesis is that as the coils get smaller the volume occupied by the copper turns increases relative to the open I.D.; as such a large proportion of the sample is brought in close proximity to the coil turns and likely gives rise to strong sample-coil magnetic coupling, which increases the signal. The applications of the planar microcoils are demonstrated on Cypselurus poecilopterus (fish) and Daphnia magna (water flea) eggs. A single D. magna egg on a 50 μm coil yielded at least 3000 times the mass sensitivity (∼9,000,000 time saving) when compared to a 5 mm probe. This value could be at least 4 times higher if the B1 homogeneity of the coils could be improved. With the current design, 80% of the signal is lost in multiple pulse experiments that rely on phase inversion and signal cancellation between scans. The data were extrapolated to predict that biological samples as small as ∼4 μm may become accessible via planar microcoil designs. To fulfill their potential for in situ metabolic screening, specialized magnetic susceptibility matched sample holders that restrict the sample to the homogeneous B1 field region (i.e. within the 90% RF field) of the coil and advanced experiments that narrow spectral lines, suppress lipids and disperse signals into multiple dimensions will be required.

The recent breakthroughs in NMR probe technologies resulted in the development of MAS NMR probes with rotation frequencies exceeding 100 kHz. Herein, we explore dramatic increases in sensitivity and resolution observed at MAS frequencies of 110-111 kHz in a novel 0.7 mm HCND probe that enable structural analysis of fully protonated biological systems. Proton-detected 2D and 3D correlation spectroscopy under such conditions requires only 0.1-0.5 mg of sample and a fraction of time compared to conventional C-13-detected experiments. We discuss the performance of several proton-and heteronuclear- (C-13-, N-15-) based correlation experiments in terms of sensitivity and resolution, using a model microcrystalline fMLF tripeptide. We demonstrate the applications of ultrafast MAS to a large, fully protonated protein assembly of the 231-residue HIV-1 CA capsid protein. Resonance assignments of protons and heteronuclei, as well as H-1-N-15 dipolar and H-1(N) CSA tensors are readily obtained from the high sensitivity and resolution proton-detected 3D experiments. The approach demonstrated here is expected to enable the determination of atomic-resolution structures of large protein assemblies, inaccessible by current methodologies.

Total correlation spectroscopy (TOCSY) is a key experiment to assign nuclear magnetic resonance (NMR) spectra of complex molecules. Carbon-13 TOCSY experiments are essential to assign signals of protein side chains. However, the performance of carbon-13 TOCSY deteriorates at high magnetic fields since the necessarily limited radiofrequency irradiation fails to cover the broad range of carbon-13 frequencies. Here, we introduce a new concept to overcome the limitations of TOCSY by using two-field NMR spectroscopy. In two-field TOCSY experiments, chemical shifts are labelled at high field but isotropic mixing is performed at a much lower magnetic field, where the frequency range of the spectrum is drastically reduced. We obtain complete correlations between all carbon-13 nuclei belonging to amino acids across the entire spectrum: aromatic, aliphatic and carboxylic. Two-field TOCSY should be a robust and general approach for the assignment of uniformly carbon-13 labelled molecules in high-field and ultra-high field NMR spectrometers beyond 1000MHz.

Nuclear magnetic resonance (NMR) spectroscopy is extremely powerful for chemical analysis but it suffers from lower mass sensitivity compared to many other analytical detection methods. NMR microcoils have been developed in response to this limitation, but interfacing these coils with small sample volumes is a challenge. We introduce here the first digital microfluidic system capable of interfacing droplets of analyte with microcoils in a high-field NMR spectrometer. A finite element simulation was performed to assist in determining appropriate system parameters. After optimization, droplets inside the spectrometer could be controlled remotely, permitting the observation of processes such as xylose-borate complexation and glucose oxidase catalysis. We propose that the combination of DMF and NMR will be a useful new tool for a wide range of applications in chemical analysis.

A series of 18 nitroxide biradicals derived from bTurea has been prepared, and their enhancement factors ɛ ((1)H) in cross-effect dynamic nuclear polarization (CE DNP) NMR experiments at 9.4 and 14.1 T and 100 K in a DNP-optimized glycerol/water matrix ("DNP juice") have been studied. We observe that ɛ ((1)H) is strongly correlated with the substituents on the polarizing agents, and its trend is discussed in terms of different molecular parameters: solubility, average e-e distance, relative orientation of the nitroxide moieties, and electron spin relaxation times. We show that too short an e-e distance or too long a T1e can dramatically limit ɛ ((1)H). Our study also shows that the molecular structure of AMUPol is not optimal and its ɛ ((1)H) could be further improved through stronger interaction with the glassy matrix and a better orientation of the TEMPO moieties. A new AMUPol derivative introduced here provides a better ɛ ((1)H) than AMUPol itself (by a factor of ca. 1.2).

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two-field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low-field dimension. Two-field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.

Comprehensive Multiphase NMR provides an overview as to all components (liquids, gels, solids) in a living organism.

Since the isolation of soil organic matter in 1786, tens of thousands of publications have searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical role in defining soil organic matter but traditional approaches remove key information such as the distribution of components at the soil-water interface and conformational information. Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural components in soil organic matter are identified to be largely composed of macromolecular inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants. Here, for the first time, a complete range of physical states of a whole soil can be studied. This provides a more detailed understanding of soil organic matter at the molecular level itself key to develop the most efficient soil remediation and agricultural techniques, and better predict carbon sequestration and climate change.

Nuclear magnetic resonance (NMR) is a ubiquitous branch of spectroscopy that can explore matter at the scale of an atom. Significant improvements in sensitivity and resolution have been driven by a steady increase of static magnetic field strengths. However, some properties of nuclei may be more favourable at low magnetic fields. For example, transverse relaxation due to chemical shift anisotropy increases sharply at higher magnetic fields leading to line-broadening and inefficient coherence transfers. Here, we present a two-field NMR spectrometer that permits the application of rf-pulses and acquisition of NMR signals in two magnetic centres. Our prototype operates at 14.1 T and 0.33 T. The main features of this system are demonstrated by novel NMR experiments, in particular a proof-of-concept correlation between zero-quantum coherences at low magnetic field and single quantum coherences at high magnetic field, so that high resolution can be achieved in both dimensions, despite a ca. 10 ppm inhomogeneity of the low-field centre. Two-field NMR spectroscopy offers the possibility to circumvent the limits of high magnetic fields, while benefiting from their exceptional sensitivity and resolution. This approach opens new avenues for NMR above 1 GHz.

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Comprehensive multiphase NMR is a novel NMR technique that permits all components (solutions, gels, and solids) to be studied in unaltered natural samples. In this study a wide range of CMP-NMR interaction and editing-based experiments are combined to follow contaminants (pentafluorophenol (PFP) and perfluorooctanoic acid (PFOA)) from the solution state (after a spill) through the gel-state and finally into the true solid-state (sequestered) in an intact water-swollen soil. Kinetics experiments monitoring each phase illustrate PFOA rapidly transfers from solution to the solid phase while for PFP the process is slower with longer residence times in the solution and gel phase. Interaction-based experiments reveal that PFOA enters the soil via its hydrophobic tails and selectively binds to soil microbial protein. PFP sorption shows less specificity exhibiting interactions with a range of gel and solid soil components with a preference toward aromatics (mainly lignin). The results indicate that in addition to more traditional measurements such as Koc, other factors including the influence of the contaminant on the soil-water interface, specific biological interactions, soil composition (content of lignin, protein, etc.) and physical accessibility/swellability of soil organic components will likely be central to better explaining and predicting the true behavior of contaminants in soil.

Environmental context Novel technology is used to examine oil contaminated soil to better understand this longstanding problem. The data indicate that oil forms a non-discriminant layer over all the soil components, which in their natural state would be exposed to water, and that it retains certain polar compounds while contributing other oil contaminants to the surrounding porewater and groundwater. Such molecular level information helps to better understand the reoccurrence of hydrophobicity in remediated soil, and could lead to novel clean-up methods. Abstract Comprehensive multiphase (CMP) NMR spectroscopy is a novel NMR technology introduced in 2012. CMP NMR spectroscopy permits the analysis of solid, gel and liquid phases in unaltered natural samples. Here the technology is applied to control and oil contaminated soils to understand the molecular processes that give rise to non-wettable soils. 13C solid-state NMR spectroscopy is found to be excellent for studying the bulk rigid components of the soils whereas 1H solution and gel-state NMR provide a complimentary overview to subtleties occurring at the soil–water interface. Considered holistically the NMR data support the finding that the oil forms a non-discriminant layer over all the soil components, which in the natural state, would be exposed to water. Specifically, the oil was found to preferentially coat aliphatics and carbohydrates that normally stick out at the soil–water interface. In addition, it was shown that the oil forms a barrier that keeps small polar molecules such as formic acid inside the soil. At the soil–water interface selective oil components, such as asphaltenes, were found to exhibit unrestricted diffusion, suggesting that these components could leach into surrounding groundwater.

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)‐NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid‐state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ . The technology is evaluated using wild‐type Arabidopsis thaliana and the cellulose‐deficient mutant ectopic lignification1 ( eli1 ) as examples. Using CMP‐NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP‐NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT. Copyright © 2015 John Wiley & Sons, Ltd.

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K+ channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle. High-field DNP thus offers atomic insight into the role of molecular plasticity during the course of biomolecular function in a complex cellular environment.