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Fluorinated drugs occupy a large and growing share of the pharmaceutical market. Here, we explore high-frequency, 60 to 111 kHz, 19F magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for the structural characterization of fluorinated active pharmaceutical ingredients in commercial formulations of seven blockbuster drugs: Celebrex, Cipro, Crestor, Levaquin, Lipitor, Prozac, and Zyvox. 19F signals can be observed in a single scan, and spectra with high signal-to-noise ratios can be acquired in minutes. 19F spectral parameters, such as chemical shifts and line widths, are sensitive to both the nature of the fluorine moiety and the formulation. We anticipate that the fast 19F MAS NMR-based approach presented here will be valuable for the rapid analysis of fluorine-containing drugs in a wide variety of formulations.

(19)F magic angle spinning (MAS) NMR spectroscopy is a powerful tool for characterization of fluorinated solids. The recent development of (19)F MAS NMR probes, operating at spinning frequencies of 60–111 kHz, enabled analysis of systems spanning from organic molecules to pharmaceutical formulations to biological assemblies, with unprecedented resolution. Herein, we systematically evaluate the benefits of high MAS frequencies (60–111 kHz) for 1D and 2D (19)F-detected experiments in two pharmaceuticals, the antimalarial drug mefloquine and a formulation of the cholesterol-lowering drug atorvastatin calcium. We demonstrate that (1)H decoupling is essential and that scalar-based, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum coherence (HMQC) correlation experiments become feasible and efficient at the MAS frequency of 100 kHz. This study opens doors for the applications of high frequency (19)F MAS NMR to a wide range of problems in chemistry and biology.

Fluorine-containing compounds comprise 20 to 30 percent of all commercial drugs, and the proportion of fluorinated pharmaceuticals is rapidly growing. While magic angle spinning (MAS) NMR spectroscopy is a popular technique for analysis of solid pharmaceutical compounds, fluorine has been underutilized as a structural probe so far. Here, we report a fast (40-60 kHz) MAS 19F NMR approach for structural characterization of fluorine-containing crystalline pharmaceutical compounds at natural abundance, using the antimalarial fluorine-containing drug mefloquine as an example. We demonstrate the utility of 2D 19F-13C and 19F-19F dipolar-coupling-based correlation experiments for 19F and 13C resonance frequency assignment, which permit identification of crystallographically inequivalent sites. The efficiency of 19F-13C cross-polarization and the effect of 1H and 19F decoupling on spectral resolution and sensitivity were evaluated in a broad range of experimental conditions. We further demonstrate a protocol for measuring accurate interfluorine distances based on 1D DANTE-RFDR experiments combined with multispin numerical simulations.

The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (N

Heteronuclear dipolar coupling is indispensable in revealing vital information related to the molecular structure and dynamics, as well as intermolecular interactions in various solid materials. Although numerous approaches have been developed to selectively reintroduce heteronuclear dipolar coupling under MAS, most of them lack universality and can only be applied to limited spin systems. Herein, we introduce a new and robust technique dubbed phase modulated rotary resonance (PMRR) for reintroducing heteronuclear dipolar couplings while suppressing all other interactions under a broad range of MAS conditions. The standard PMRR requires the radiofrequency (RF) field strength of only twice the MAS frequency, can efficiently recouple the dipolar couplings with a large scaling factor of 0.50, and is robust to experimental imperfections. Moreover, the adjustable window modification of PMRR, dubbed wPMRR, can improve its performance remarkably, making it well suited for the accurate determination of dipolar couplings in various spin systems. The robust performance of such pulse sequences has been verified theoretically and experimentally via model compounds, at different MAS frequencies. The application of the PMRR technique was demonstrated on the H-ZSM-5 zeolite, where the interaction between the Bronsted acidic hydroxyl groups of H-ZSM-5 and the absorbed trimethylphosphine oxide (TMPO) were probed, revealing the detailed configuration of super acid sites.

HIV-1 capsid plays multiple key roles in viral replication, and inhibition of capsid assembly is an attractive target for therapeutic intervention. Here, we report the atomic-resolution structure of capsid protein (CA) tubes, determined by magic-angle spinning NMR and data-guided molecular dynamics simulations. Functionally important regions, including the NTD β-hairpin, the cyclophilin A-binding loop, residues in the hexamer central pore, and the NTD-CTD linker region, are well defined. The structure of individual CA chains, their arrangement in the pseudo-hexameric units of the tube and the inter-hexamer interfaces are consistent with those in intact capsids and substantially different from the organization in crystal structures, which feature flat hexamers. The inherent curvature in the CA tubes is controlled by conformational variability of residues in the linker region and of dimer and trimer interfaces. The present structure reveals atomic-level detail in capsid architecture and provides important guidance for the design of novel capsid inhibitors. Structures of HIV-1 capsid protein (CA) in tubular assemblies, determined by MAS-NMR, reveal the basis of CA’s conformational plasticity.

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein–protein or protein–ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as ((1)H)-(77)Se-(13)C double cross polarization or {(77)Se}-(13)C REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that (77)Se-(13)C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is ~ 5 Å, but longer range distances up to ~ 7 Å can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein–protein or protein–ligand binding interfaces.

Fluorination is a versatile and valuable modification for numerous systems, and (19)F NMR spectroscopy is the premier method for their structural characterization. (19)F chemical shift anisotropy is a sensitive probe of structure and dynamics, even though (19)F chemical shift tensors have been reported for only a handful of systems to date. Here, we explore γ-encoded R-symmetry based recoupling sequences for the determination of (19)F chemical shift tensors in fully protonated organic solids at high, 60–100 kHz MAS frequencies. We show that the performance of (19)F-RNCSA experiments improves with increasing MAS frequencies, and that (1)H decoupling is required to determine accurate chemical shift tensor parameters. In addition, these sequences are tolerant to B(1)-field inhomogeneity making them suitable for a wide range of systems and experimental conditions.

Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility, and cell division. Two motors, kinesin and cytoplasmic dynein link the MT network to transported cargos using ATP for force generation. Here, we report an all-atom NMR structure of nucleotide-free kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure reveals the position and orientation of the functionally important neck linker and how ADP induces structural and dynamic changes that ensue in the neck linker. These results demonstrate that the neck linker is in the undocked conformation and oriented in the direction opposite to the KIF5B movement. Chemical shift perturbations and intensity changes indicate that a significant portion of ADP-KIF5B is in the neck linker docked state. This study also highlights the unique capability of MAS NMR to provide atomic-level information on dynamic regions of biological assemblies.

We report remarkably high, up to 100-fold, signal enhancements in (19)F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 CA capsid protein assemblies. These enhancements correspond to absolute sensitivity ratios of 12–29 and are of similar magnitude as seen for (1)H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D (19)F-(13)C 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 (19)F 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.

Fluorine-containing compounds comprise twenty to thirty percent of all commercial drugs, and the proportion of fluorinated pharmaceuticals is rapidly growing. While magic angle spinning (MAS) NMR spectroscopy is a popular technique for analysis of solid pharmaceutical compounds, fluorine has been underutilized as a structural probe so far. Here we report a fast (40 kHz) MAS (19)F NMR approach for structural characterization of fluorine-containing crystalline pharmaceutical compounds at natural abundance, using the antimalarial fluorine-containing drug mefloquine as an example. We demonstrate the utility of 2D (19)F-(13)C and (19)F-(19)F dipolar-coupling-based correlation experiments for (19)F and (13)C resonance frequency assignment, which permit identification of crystallographically inequivalent sites. The efficiency of (19)F-(13)C cross-polarization (CP) as well as the effect of (1)H and (19)F decoupling on spectral resolution and sensitivity were evaluated in a broad range of experimental conditions. We further demonstrate a protocol for measuring accurate interfluorine distances based on 1D DANTE-RFDR experiments combined with multi-spin numerical simulations.

Dynamic nuclear polarization-enhanced (DNP) magic angle spinning (MAS) NMR of biological systems is a rapidly growing field. Large signal enhancements make the technique particularly attractive for signal-limited cases, such as studies of complex biological assemblies or at natural isotopic abundance. However, spectral resolution is considerably reduced compared to ambient-temperature non-DNP spectra. Herein, we report a systematic investigation into sensitivity and resolution of 1D and 2D 13C-detected DNP MAS NMR experiments on HIV-1 CA tubular assemblies. We show that the magnitude and sign of signal enhancement as well as the homogeneous line width are strongly dependent on the biradical concentration, the dominant polarization transfer pathway, and the enhancement buildup time. Our findings provide guidance for optimal choice of sample preparation and experimental conditions in DNP experiments.

In this chapter, we discuss recent advances in solid-state magic angle spinning (MAS) NMR spectroscopy of microcrystalline proteins. We review approaches for isotopic labelling and microcrystalline sample preparation and multidimensional MAS NMR correlation experiments for resonance assignment and distance restraint determination as well as the measurement of chemical shift and dipolar tensors. These experiments include heteronuclear 13C/15N-, 1H- and 19F- based techniques in the regimes of moderate (less than 60 kHz) as well as high (greater than 60 kHz) MAS frequencies. We present examples of applications of these techniques to determine 3D structures of proteins and their interactions with water, small-molecule ligands, other proteins as well as nucleic acids. The recent advances in high field magnet and MAS NMR probe technologies open new avenues for methods development and applications to larger proteins and biological assemblies intractable by conventional methods.

19 F 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) 19 F 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 19 F 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 19 F-19 F correlations, yielding interatomic distances of the order of 20 A. Our results demonstrate the potential of fast MAS 19 F NMR spectroscopy for structural analysis in large biological assemblies.

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

Hydrocracking is an insufficiently explored route for chemical recycling of plastics waste. Platinum tungstated zirconia (Pt-WZr) was used in a batch reactor at low temperatures of 250 °C and 30 bar H2 pressure for 1−24 h reaction times using low-density polyethylene (LDPE, Mw∼76 kDa). We find Pt-WZr is a bifunctional catalyst for LDPE hydrocracking leading to higher value branched fuel- and lubricant-ranged alkanes. We demonstrate that the catalyst metal-to-acid site molar ratio (MAB) shifts the product distribution to larger cracked products and increases the isomerization degree in the residual polymer. We propose a new adhesive isomerization mechanism between the metal and Bronsted acid sites in parallel with slow polymer chain cracking, caused by competitive adsorption of the polymer over the liquid products and stereochemical hindrance of methines. This study provides a blueprint on how to engineer effective catalysts for hydrocracking polyolefin plastic wastes using the MAB as a catalyst descriptor.

Chemical shifts are highly sensitive probes of local conformation and overall structure. Both isotropic shifts and chemical shift tensors are readily accessible from NMR experiments but their quantum mechanical calculations remain challenging. In this work, we report and compare accurately measured and calculated (15)N(H) and (13)C(α) chemical shift tensors in proteins, using the microcrystalline agglutinin from Oscillatoria agardhii (OAA). Experimental (13)C(α) and (15)N(H) chemical tensors were obtained by solid-state NMR spectroscopy, employing tailored recoupling sequences, and for their quantum mechanics/molecular mechanics (QM/MM) calculations different sets of functionals were evaluated. We show that (13)C(α) chemical shift tensors are primarily determined by backbone dihedral angles and dynamics, while (15)N(H) tensors mainly depend on local electrostatic contributions from solvation and hydrogen bonding. In addition, the influence of including crystallographic waters, the molecular mechanics geometry optimization protocol, and the level of theory on the accuracy of the calculated chemical shift tensors is discussed. Specifically, the power of QM/MM calculations in accurately predicting the unusually upfield shifted (1)H(N) G26 and G93 resonances is highlighted. Our integrated approach is expected to benefit structure refinement of proteins and protein assemblies.

Long-range interatomic distance restraints are critical for the determination of molecular structures by NMR spectroscopy, both in solution and in the solid state. Fluorine is a powerful NMR probe in a wide variety of contexts, owing to its favorable magnetic properties, ease of incorporation into biological molecules, and ubiquitous use in synthetic organic molecules designed for diverse applications. Due to the large gyromagnetic ratio of the 100% naturally abundant (19)F isotope, interfluorine distances as long as 20 Å are accessible in magic angle spinning (MAS) dipolar recoupling experiments. Herein, we present an approach for the determination of accurate interfluorine distances in multi-spin systems, using the finite pulse RFDR (fpRFDR) at high MAS frequencies of 40–60 kHz. We use a series of crystalline “molecular ruler” solids, difluorobenzoic acids and 7F-L-tryptophan, for which the intra- and intermolecular interfluorine distances are known. We describe the optimal experimental conditions for accurate distance determinations, including the choice of a phase cycle, the relative advantages of selective inversion 1D vs. 2D correlation experiments, and the appropriate numerical simulation protocols. A best strategy for the analysis of RFDR exchange curves in organic solids with extended spin interaction networks is presented, which, even in the absence of crystal structures, can be potentially incorporated into NMR structure determination.

We report 43 Ca and 13 C solid-state nuclear magnetic resonance (NMR) spectroscopic studies of the ethylene glycol solvate of atorvastatin calcium. The 13 C and 43 Ca chemical shift and 43 Ca quadrupolar coupling tensor parameters are reported. The results are interpreted in terms of the reported X-ray diffraction crystal structure of the solvate and are compared with the NMR parameters of atorvastatin calcium trihydrate, the active pharmaceutical ingredient in Lipitor®. Hartree-Fock and density functional theory calculations of the NMR parameters based on a cluster model derived from the optimized X-ray diffraction crystal structure of the ethylene glycol solvate of atorvastatin calcium are in reasonable agreement with the experimental 43 Ca and 13 C NMR measurables.

We report

We present a systematic investigation into the attainable accuracy and precision of protein structures determined by heteronuclear magic angle spinning solid-state NMR for a set of four proteins of varied size and secondary structure content. Structures were calculated using synthetically generated random sets of C-C distances up to 7 A at different degrees of completeness. For single-domain proteins, 9–15 restraints per residue are sufficient to derive an accurate model structure, while maximum accuracy and precision are reached with over 15 restraints per residue. For multi-domain proteins and protein assemblies, additional information on domain orientations, quaternary structure and/or protein shape is needed. As demonstrated for the HIV-1 capsid protein assembly, this can be accomplished by integrating MAS NMR with cryoEM data. In all cases, inclusion of TALOS-derived backbone torsion angles improves the accuracy for small number of restraints, while no further increases are noted for restraint completeness above 40%. In contrast, inclusion of TALOS-derived torsion angle restraints consistently increases the precision of the structural ensemble at all degrees of distance restraint completeness.

(19)F 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) (19)F solid-state NMR in assemblies of HIV-1 capsid protein. Tryptophan residues with fluorine substitutions in the 5 position of the indole ring were used as the reporters. The (19)F chemical shifts for the five tryptophans 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 (19)F-(19)F correlations, yielding interatomic distances of the order of 20 Å. To our knowledge, this is the first example of (19)F MAS NMR being applied to a very large protein assembly. Our results demonstrate the potential of fast MAS (19)F NMR for structural analysis in large biological assemblies.

The (19)F 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 (DFT). Significant narrowing of the (19)F lines was observed under fast MAS conditions, at spinning frequencies above 50 kHz. The parameters characterizing the (19)F 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 (19)F 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 (19)F-based MAS NMR structural analysis of proteins and protein assemblies.

The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In the recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

As a result of profound gains in sensitivity and resolution afforded by ultrahigh magnetic fields, transformative applications in the fields of structural biology and materials science are being realized. The development of dual low temperature superconducting (LTS)/high-temperature superconducting (HTS) magnets has enabled the achievement of magnetic fields above 1 GHz (23.5 T), which will open doors to an unprecedented new range of applications. In this contribution, we discuss the promise of ultrahigh field magnetic resonance. We highlight several methodological developments pertinent at high-magnetic fields including measurement of 1H-1H distances and 1H chemical shift anisotropy in the solid state as well as studies of quadrupolar nuclei such as 17O. Higher magnetic fields have advanced heteronuclear detection in solution NMR, valuable for applications including metabolomics and disordered proteins, as well as expanded use of proton detection in the solid state in conjunction with ultrafast magic angle spinning. We also present several recent applications to structural studies of the AP205 bacteriophage, the M2 channel from Influenza A, and biomaterials such as human bone. Gains in sensitivity and resolution from increased field strengths will enable advanced applications of NMR spectroscopy including in vivo studies of whole cells and intact virions.

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA–SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA–SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix–coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions., The process of HIV particle maturation involves complex molecular transitions. Here the authors combine NMR spectroscopy, cryo-EM, and molecular dynamics simulations to provide insight into the conformational equilibria in CA-SP1 assemblies relevant to HIV-1 maturation intermediates formation.

International audience; 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 13C-detected experiments. We discuss the performance of several proton- and heteronuclear- (13C-,15N-) 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 1H-15N dipolar and 1HN 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.

In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.

NMR chemical shifts are exquisitely sensitive probes for conformation and dynamics in molecules and supramolecular assemblies. Although isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, the 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations for analysis of 13Cα and 15NH isotropic chemical shifts. The influence of local interactions, quaternary contacts, and dynamics on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies.

We demonstrate the determination of quantitative rates of molecular reorientation in the solid state with rotating frame (R1ρ) relaxation measurements. Reorientation of the carbon chemical shift anisotropy (CSA) tensor was used to probe site-specific conformational exchange in a model system, d6-dimethyl sulfone (d6-DMS). The CSA as a probe of exchange has the advantage that it can still be utilized when there is no dipolar mechanism (i.e. no protons attached to the site of interest). Other works have presented R1ρ measurements as a general indicator of dynamics, but this study extracts quantitative rates of molecular reorientation from the R1ρ values. Some challenges of this technique include precise knowledge of sample temperature and determining the R 2 0 contribution to the observed relaxation rate from interactions other than molecular reorientation, such as residual dipolar couplings or fast timescale dynamics; determination of this term is necessary in order to quantify the exchange rate due to covariance between the 2 terms. Low-temperature experiments measured an R 2 0 value of 1.8 ± 0.2 s−1 Allowing for an additional relaxation term ( R 2 0 ), which was modeled as both temperature-dependent and temperature-independent, rates of molecular reorientation were extracted from field strength-dependent R1ρ measurements at four different temperatures and the activation energy was determined from these exchange rates. The activation energies determined were 74.7 ± 4.3 kJ/mol and 71.7 ± 2.9 kJ/mol for the temperature-independent and temperature-dependent R 2 0 models respectively, in excellent agreement with literature values. The results of this study suggest important methodological considerations for the application of the method to more complicated systems such as proteins, such as the importance of deuterating samples and the need to make assumptions regarding the R 2 0 contribution to relaxation.

Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.

We demonstrate the determination of quantitative rates of molecular reorientation in the solid state with rotating frame (R(1ρ)) relaxation measurements. Reorientation of the carbon chemical shift anisotropy (CSA) tensor was used to probe site-specific conformational exchange in a model system, d(6)-dimethyl sulfone (d(6)-DMS). The CSA as a probe of exchange has the advantage that it can still be utilized when there is no dipolar mechanism (i.e. no protons attached to the site of interest). Other works have presented R(1ρ) measurements as a general indicator of dynamics, but this study extracts quantitative rates of molecular reorientation from the R(1ρ) values. Some challenges of this technique include precise knowledge of sample temperature and determining the R(2)(0) contribution to the observed relaxation rate from interactions other than molecular reorientation, such as residual dipolar couplings or fast timescale dynamics; determination of this term is necessary in order to quantify the exchange rate due to covariance between the 2 terms. Low-temperature experiments measured an R(2)(0) value of 1.8±0.2s(-1) Allowing for an additional relaxation term (R(2)(0)), which was modeled as both temperature-dependent and temperature-independent, rates of molecular reorientation were extracted from field strength-dependent R(1ρ) measurements at four different temperatures and the activation energy was determined from these exchange rates. The activation energies determined were 74.7±4.3kJ/mol and 71.7±2.9kJ/mol for the temperature-independent and temperature-dependent R(2)(0) models respectively, in excellent agreement with literature values. The results of this study suggest important methodological considerations for the application of the method to more complicated systems such as proteins, such as the importance of deuterating samples and the need to make assumptions regarding the R(2)(0) contribution to relaxation.

A new application of solid-state rotating frame (R 1ρ) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R 1ρ relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d 6 -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins could represent a new site-specific probe of conformational dynamics.

Despite breakthroughs in MAS NMR hardware and experimental methodologies sensitivity remains a major challenge for large and complex biological systems. Here, we report 3–4 fold higher sensitivities obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments for large assemblies that are otherwise time-prohibitive, such as 2D (15)N-(15)N proton-driven spin diffusion and (15)N-(13)C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins - demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of applications, especially for biological systems suffering from low intrinsic sensitivity and at physiological temperatures.