Integrating 19F Distance Restraints for Accurate Protein Structure Determination by Magic Angle Spinning NMR Spectroscopy.

Traditional protein structure determination by magic angle spinning (MAS) solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from 13C-, 15N-, and 1H-based dipolar-based correlation experiments. Here, we show that 19F fast (60 kHz) MAS NMR spectroscopy can supply additional, longer distances. Using 4F-Trp,U-13C,15N crystalline Oscillatoria agardhii agglutinin (OAA), we demonstrate that judiciously designed 2D and 3D 19F-based dipolar correlation experiments such as (H)CF, (H)CHF, and FF can yield interatomic distances in the 8-16 Å range. Incorporation of fluorine-based restraints into structure calculation improved the precision of Trp side chain conformations as well as regions in the protein around the fluorine containing residues, with notable improvements observed for residues in proximity to the Trp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops, which lacked sufficient long-range 13C-13C distance restraints. Our work highlights the use of fluorine and 19F fast MAS NMR spectroscopy as a powerful structural biology tool.

Spatial Organization of Lipid Nanoparticle siRNA Delivery Systems Revealed by an Integrated Magnetic Resonance Approach.

Lipid nanoparticles (LNPs) are increasingly finding applications in targeted drug delivery, including for subcutaneous, intravenous, inhalation, and vaccine administration. While a variety of microscopy techniques are widely used for LNP characterization, their resolution does not allow for characterization of the spatial organization of different components, such as the excipients, targeting agents, or even the active ingredient. Herein, an approach is presented to probe the spatial organization of individual constituent groups of LNPs used for siRNA-based drug delivery, currently in clinical trials, by multinuclear solid-state magic-angle-spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Dynamic nuclear polarization is exploited (DNP) for sensitivity enhancement, together with judicious 2H labeing, to detect functionally important LNP constituents, the siRNA and the targeting agent (<1-2 w/v%), respectively, and achieve a structural model of the LNP locating the siRNA in the core, the targeting agent below the surface, and the sugars above the lipid bilayer at the surface. The integrated approach presented here is applicable for structural analysis of LNPs and can be extended more generally to other multi-component biological formulations.

19F Fast Magic-Angle Spinning NMR Spectroscopy on Microcrystalline Complexes of Fluorinated Ligands and the Carbohydrate Recognition Domain of Galectin-3.

Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast 19F- and 1H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D 13C-19F and 1H-19F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that 19F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.

Total and Class-Specific Determination of Fluorinated Compounds in Consumer and Food Packaging Samples Using Fluorine-19 Solid-State Nuclear Magnetic Resonance Spectroscopy.

Hamburger wrapping paper, coated with water-based barrier coatings, used in the food packaging industry was studied by using the total organic fluorine (TOF) method based on combustion ion chromatography and fluorine-19 solid-state nuclear magnetic resonance (19F ss-NMR) spectroscopy. Although the TOF method is a fast and affordable method used to screen for per- and polyfluoroalkyl substances (PFAS), the amount of fluorine it measures is heavily dependent on the extraction step and, therefore could lead to inaccurate results. Fluorine-19 ss-NMR spectroscopy can differentiate between organic and inorganic fluorinated sources, eliminating the need for sample clean up. To illustrate this, the 19F ss-NMR spectra of clean coated paper samples that contained naturally occurring F- ions from the talc raw material and spiked samples containing perfluorooctanoic acid were compared. A range of experimental conditions was explored to improve sensitivity for low PFAS concentrations (in the order of 10-20 mg/kg). Despite the disadvantages of ss-NMR spectroscopy, such as the low limit of detection and resolution, the results demonstrate it can be a viable tool to directly detect PFAS moieties in consumer and food packaging. Therefore, 19F solid-state NMR spectroscopy challenges and complements current methods, which only provide indirect evidence of the presence of PFAS.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Tuning the reactivity of carbon surfaces with oxygen-containing functional groups.

Oxygen-containing carbons are promising supports and metal-free catalysts for many reactions. However, distinguishing the role of various oxygen functional groups and quantifying and tuning each functionality is still difficult. Here we investigate the role of Brønsted acidic oxygen-containing functional groups by synthesizing a diverse library of materials. By combining acid-catalyzed elimination probe chemistry, comprehensive surface characterizations, 15N isotopically labeled acetonitrile adsorption coupled with magic-angle spinning nuclear magnetic resonance, machine learning, and density-functional theory calculations, we demonstrate that phenolic is the main acid site in gas-phase chemistries and unexpectedly carboxylic groups are much less acidic than phenolic groups in the graphitized mesoporous carbon due to electron density delocalization induced by the aromatic rings of graphitic carbon. The methodology can identify acidic sites in oxygenated carbon materials in solid acid catalyst-driven chemistry.

Structural basis of HIV-1 maturation inhibitor binding and activity.

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

Magic-angle-spinning NMR structure of the kinesin-1 motor domain assembled with microtubules reveals the elusive neck linker orientation.

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.

19F fast MAS (60-111 kHz) dipolar and scalar based correlation spectroscopy of organic molecules and pharmaceutical formulations.

19F magic angle spinning (MAS) NMR spectroscopy is a powerful tool for characterization of fluorinated solids. The recent development of 19F 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 19F-detected experiments in two pharmaceuticals, the antimalarial drug mefloquine and a formulation of the cholesterol-lowering drug atorvastatin calcium. We demonstrate that 1H 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 19F MAS NMR to a wide range of problems in chemistry and biology.

Electronic modulation of metal-support interactions improves polypropylene hydrogenolysis over ruthenium catalysts.

Ruthenium (Ru) is the one of the most promising catalysts for polyolefin hydrogenolysis. Its performance varies widely with the support, but the reasons remain unknown. Here, we introduce a simple synthetic strategy (using ammonia as a modulator) to tune metal-support interactions and apply it to Ru deposited on titania (TiO2). We demonstrate that combining deuterium nuclear magnetic resonance spectroscopy with temperature variation and density functional theory can reveal the complex nature, binding strength, and H amount. H2 activation occurs heterolytically, leading to a hydride on Ru, an H+ on the nearest oxygen, and a partially positively charged Ru. This leads to partial reduction of TiO2 and high coverages of H for spillover, showcasing a threefold increase in hydrogenolysis rates. This result points to the key role of the surface hydrogen coverage in improving hydrogenolysis catalyst performance.

A systematic review and meta-analysis of the evidence for community-based HIV testing on men's engagement in the HIV care cascade.

OBJECTIVE: Men with HIV are less likely than women to know their status, be on antiretroviral therapy, and be virally suppressed. This review examined men's community-based HIV testing services (CB-HTS) outcomes. DESIGN: Systematic review and meta-analysis. METHODS: We searched seven databases and conference abstracts through July 2018. We estimated pooled proportions and/or risk ratios (for meta-analyses) for each outcome using random effects models. RESULTS: 188 studies met inclusion criteria. Common testing models included targeted outreach (e.g. mobile testing), home-based testing, and testing at stand-alone community sites. Across 25 studies reporting uptake, 81% (CI: 75-86%) of men offered testing accepted it. Uptake was higher among men reached through CB-HTS than facility-based HTS (RR = 1.39; CI: 1.13-1.71). Over 69% (CI: 64-71%) of those tested through CB-HTS were men, across 184 studies. Across studies reporting new HIV-positivity among men (n = 18), 96% were newly diagnosed (CI: 77-100%). Across studies reporting linkage to HIV care (n = 8), 70% (CI: 36-103%) of men were linked to care. Across 57 studies reporting sex-disaggregated data for CB-HTS conducted among key populations, men's uptake was high (80%; CI: 70-88%) and nearly all were newly diagnosed and linked to care (95%; CI: 94-100%; and 94%; CI: 88-100%, respectively). CONCLUSION: CB-HTS is an important strategy for reaching undiagnosed men with HIV from the general population and key population groups, particularly using targeted outreach models. When compared to facility-based HIV testing services, men tested through CB-HTS are more likely to uptake testing, and nearly all men who tested positive through CB-HTS were newly diagnosed. Linkage to care may be a challenge following CB-HTS, and greater efforts and research are needed to effectively implement testing strategies that facilitate rapid ART initiation and linkage to prevention services.

Determination of accurate 19F chemical shift tensors with R-symmetry recoupling at high MAS frequencies (60-100 kHz).

Fluorination is a versatile and valuable modification for numerous systems, and 19F NMR spectroscopy is the premier method for their structural characterization. 19F chemical shift anisotropy is a sensitive probe of structure and dynamics, even though 19F 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 19F chemical shift tensors in fully protonated organic solids at high, 60-100 kHz MAS frequencies. We show that the performance of 19F-RNCSA experiments improves with increasing MAS frequencies, and that 1H decoupling is required to determine accurate chemical shift tensor parameters. In addition, these sequences are tolerant to B1-field inhomogeneity making them suitable for a wide range of systems and experimental conditions.

Atomic-Resolution Structure of SARS-CoV-2 Nucleocapsid Protein N-Terminal Domain.

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 (NNTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline NNTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, ß-hairpin loop. In addition, ultrafast (100 kHz) MAS 1H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.

77Se-13C based dipolar correlation experiments to map selenium sites in microcrystalline proteins.

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 (1H)-77Se-13C double cross polarization or {77Se}-13C 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 77Se-13C 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.

Contraception values and preferences of people living with HIV: A systematic review.

OBJECTIVE: Attention to the contraception values and preferences of those living with HIV is essential to meeting their reproductive rights and health needs. We systematically reviewed the literature on contraception values and preferences among women and men living with HIV. STUDY DESIGN: We searched ten electronic databases for articles from 1 January 2005 through 27 July 2020 for qualitative and quantitative studies of the values and preferences for contraceptive methods among individuals living with HIV. RESULTS: Twenty-one studies, primarily from sub-Saharan Africa, met the inclusion criteria. Contraception values and preferences were shaped by several factors: availability, accessibility, and convenience; perceived effectiveness; safety and tolerability; dual protection; fertility desires; partnership dynamics; and provider recommendations. Male condoms were a frequently preferred contraceptive method, offering an affordable and accessible form of dual protection against HIV and unwanted pregnancy. Fears of infertility and side effects decreased interest in hormonal contraceptive methods. Financial burdens incurred by HIV management and a desire to reduce dual reproductive health and HIV care burdens influenced preferences. Healthcare providers contributed to contraceptive preferences of women living with HIV, informing perceptions of safety, tolerability, and effectiveness. CONCLUSION: Contraception values and preferences among women living with HIV are complex and influenced by factors related and unrelated to their HIV status. Considering contraception values and preferences of people living with HIV will ensure that their autonomy and right to make decisions about the contraceptive methods best for them are upheld.

Determination of Histidine Protonation States in Proteins by Fast Magic Angle Spinning NMR.

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40-62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)-based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.

Accurate heteronuclear distance measurements at all magic-angle spinning frequencies in solid-state NMR spectroscopy.

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 Brønsted acidic hydroxyl groups of H-ZSM-5 and the absorbed trimethylphosphine oxide (TMPO) were probed, revealing the detailed configuration of super acid sites.

Fast 19F Magic-Angle Spinning Nuclear Magnetic Resonance for the Structural Characterization of Active Pharmaceutical Ingredients in Blockbuster Drugs.

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.

Adolescents and age of consent to HIV testing: an updated review of national policies in sub-Saharan Africa.

OBJECTIVES: In sub-Saharan Africa (SSA) where HIV burden is highest, access to testing, a key entry point for prevention and treatment, remains low for adolescents (aged 10-19). Access may be hampered by policies requiring parental consent for adolescents to receive HIV testing services (HTS). In 2013, the WHO recommended countries to review HTS age of consent policies. Here, we investigate country progress and policies on age of consent for HIV testing. DESIGN: Comprehensive policy review. DATA SOURCES: Policies addressing HTS were obtained through searching WHO repositories and governmental and non-governmental websites and consulting country and regional experts. ELIGIBILITY CRITERIA: HTS policies published by SSA governments before 2019 that included age of consent. DATA EXTRACTION AND SYNTHESIS: Data were extracted on HTS age of consent including exceptions based on risk and maturity. Descriptive analyses of included policies were disaggregated by Eastern and Southern Africa (ESA) and Western and Central Africa (WCA) subregions. RESULTS: Thirty-nine policies were reviewed, 38 were eligible; 19/38 (50%) permitted HTS for adolescents ≤16 years old without parental consent. Of these, six allowed HTS at ≥12 years old, two at ≥13, two at ≥14, five at ≥15 and four at ≥16. In ESA, 71% (n=15/21) allowed those of ≤16 years old to access HTS, while only 24% (n=6/25) of WCA countries allowed the same. Maturity exceptions including marriage, sexual activity, pregnancy or key population were identified in 18 policies. In 2019, 63% (n=19/30) of policies with clear age-based criteria allowed adolescents of 12-16 years old to access HIV testing without parental consent, an increase from 37% (n=14/38) in 2013. CONCLUSIONS: While many countries in SSA have revised their HTS policies, many do not specify age of consent. Revision of SSA consent to HTS policies, particularly in WCA, remains a priority to achieve the 2025 goal of 95% of people with HIV knowing their status.

Competing Transfer Pathways in Direct and Indirect Dynamic Nuclear Polarization MAS NMR Experiments on HIV-1 Capsid Assemblies: Implications for Sensitivity and Resolution.

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.