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The envelope (E) proteins of coronaviruses (CoVs) form cation‐conducting channels that are associated with the pathogenicity of these viruses. To date, high‐resolution structural information about these viroporins is limited to the SARS‐CoV E protein. To broaden our structural knowledge of other members of this family of viroporins, we now investigate the conformation of the E protein of the human coronavirus (hCoV), NL63. Using two‐ and three‐dimensional magic‐angle‐spinning NMR, we have measured 13C and 15N chemical shifts of the transmembrane domain of E (ETM), which yielded backbone (ϕ, ψ) torsion angles. We further measured the water accessibility of NL63 ETM at neutral pH versus acidic pH in the presence of Ca2+ ions. These data show that NL63 ETM adopts a regular α‐helical conformation that is unaffected by pH and the N‐terminal ectodomain. Interestingly, the water accessibility of NL63 ETM increases only modestly at acidic pH in the presence of Ca2+ compared to neutral pH, in contrast to SARS ETM, which becomes much more hydrated at acidic pH. This difference suggests a structural basis for the weaker channel conductance of α‐CoV compared to β‐CoV E proteins. The weaker E channel activity may in turn contribute to the reduced virulence of hCoV‐NL63 compared to SARS‐CoV viruses. [ABSTRACT FROM AUTHOR]

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphos-phorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy. [ABSTRACT FROM AUTHOR]

Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.

In this work, several exact and approximate analytical solutions to the quantum master equation are derived using both classical and non-classical coupling models to describe the kinetics of Hartmann-Hahn cross-polarization (HHCP) and multiple-contact CP (MCCP). Moreover, the analytical solution originally obtained by Naito and McDowell [J. Chem. Phys. 84 (1986) 4181.] is shown to be incorrect and the different regimes of spin diffusion and T1ρ relaxation are characterized by the amplitude of the second stage of the HHCP dynamics and the HHCP/MCCP crossing time. The analysis of the 1H–13C HHCP and MCCP dynamics together with (Lee-Goldburg) 1H T1ρ relaxation experimental data provides a consistent picture of spin dynamics in solid alanine and explains the apparent discrepancies previously observed between T1ρ and T1 relaxation measurements. The CH and CH3 protons relax as expected via spin diffusion towards the NH3 protons but the assumption of common proton spin temperature, in which the bottleneck of relaxation is at the NH3 sites, generally valid for T1 relaxation breaks down for T1ρ relaxation. A diffusion-limited situation in which nuclear Zeeman energy is transferred to the lattice faster than can be supplied by spin diffusion is observed instead.

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Current microcoil probe technology has emerged as a significant advancement in NMR applications to biofluids research. It has continued to excel as a hyphenated tool with other prominent microdevices, opening many new possibilities in multiple omics fields. However, this does not hold for biological samples such as intact tissue or organisms, due to the considerable challenges of incorporating the microcoil in a magic‐angle spinning (MAS) probe without relinquishing the high‐resolution spectral data. Not until 2012 did a microcoil MAS probe show promise in profiling the metabolome in a submilligram tissue biopsy with spectral resolution on par with conventional high‐resolution MAS (HR‐MAS) NMR. This result subsequently triggered a great interest in the possibility of NMR analysis with microgram tissues and striving toward the probe development of "high‐resolution" capable microcoil MAS NMR spectroscopy. This review gives an overview of the issues and challenges in the probe development and summarizes the advancements toward metabolomics. [ABSTRACT FROM AUTHOR]

[Display omitted] • Tetrakis(trimethylsilyl)silane as a standard sample for magic-angle spinning. • Sensitivity to shimming and magic-angle setting in comparison to adamantane and KBr. • Evaluation of suitability as a sensitivity standard. • Chemical shifts compared to adamantane for use as a chemical shift standard. The development of magic angle spinning (MAS) at rates ranging from 30 kHz to greater than 100 kHz has substantially advanced solid-state nuclear magnetic resonance (SSNMR) spectroscopy 1H-detection methods. The small rotors required for such MAS rates have a limited sample volume and low 13C-detection sensitivity, rendering the traditional set of standard compounds for SSNMR insufficient or highly inconvenient for shimming and magic-angle calibration. Additionally, the reproducibility of magic angle setting, chemical shift referencing, and probe position can be especially critical for SSNMR experiments at high fields. These conditions suggest the need for a high signal-to-noise ratio (SNR) 1H-detection standard compound, which is preferably multi-purpose, to simplify instrument set up for ultra-fast MAS SSNMR instruments at high magnetic fields. In this study, we present the results for setting magic angle and shimming using tetrakis(trimethylsilyl)silane (TTMSS, or TKS), a tetramethylsilane (TMS) analogue, at near 40 kHz and demonstrate that we can achieve favorable results in less time but with equal or superior precision as traditional KBr and adamantane standards. The high SNR and TMS-like chemical shift of TKS also opens the possibilities for using TKS as an internal standard with biological samples. A single rotor containing a four-component mixture of TKS, adamantane, uniformly 13C, 15N-labeled N-acetyl valine and KBr was used to perform a complete configuration and calibration of a SSNMR probe without sample changes. We anticipate TKS as a standard compound to be especially effective at very high MAS conditions and to greatly simplify the instrument set up for high and ultra-high field SSNMR instruments. [ABSTRACT FROM AUTHOR]

Among the many natural biomaterials for which information on atomic-level structure and reorientational motion can offer essential clues to function, insoluble multi-component composites with limited degrees of order are among the most challenging to study. Despite its limited sensitivity, solid-state NMR (ssNMR) is often the technique of choice to ferret out these details in carbon- and nitrogen-rich materials: this spectroscopic approach can probe many biomaterials in their native or near-native states, either with or without the introduction of stable NMR-active isotopes, or with the assistance of dynamic nuclear polarization technology. During a span of close to four decades, such research targets and ssNMR approaches have been exemplified by insects, a diverse and evolutionarily agile group of organisms with global impacts that include ecology, agriculture, and human disease. In this short review, we present case studies on insect cuticles that range from protective exoskeletons and egg capsules to the wing structures that enable flight and showcase nature's awe-inspiring beauty, highlighting the use of ssNMR spectroscopy to profile chemical composition, elucidate macromolecular architecture, and monitor metabolic development in these fascinating biological assemblies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

[Display omitted] • Demonstration of temperature impact on cross polarization stability. • Use of thermopad attenuators to reduce the effect of temperature drift. • Influence of tangent ramp parameters on cross polarization efficiency and stability. • Application to microcrystalline, membrane, and fibrous proteins. Magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful and versatile technique for probing structure and dynamics in large, insoluble biological systems at atomic resolution. With many recent advances in instrumentation and polarization methods, technology development in SSNMR remains an active area of research and presents opportunities to further improve data collection, processing, and analysis of samples with low sensitivity and complex tertiary and quaternary structures. SSNMR spectra are often collected as multidimensional data, requiring stable experimental conditions to minimize signal fluctuations (t 1 noise). In this work, we examine the factors adversely affecting signal stability as well as strategies used to mitigate them, considering laboratory environmental requirements, configuration of amplifiers, and pulse sequence parameter selection. We show that Thermopad® temperature variable attenuators (TVAs) can partially compensate for the changes in amplifier output power as a function of temperature and thereby ameliorate one significant source of instability for some spectrometers and pulse sequences. We also consider the selection of tangent ramped cross polarization (CP) waveform shapes, to balance the requirements of sensitivity and instrumental stability. These findings collectively enable improved stability and overall performance for CP-based multidimensional spectra of microcrystalline, membrane, and fibrous proteins performed at multiple magnetic field strengths. [ABSTRACT FROM AUTHOR]

Non‐invasive and real‐time recording of processes in living cells has been limited to detection of small cellular components such as soluble proteins and metabolites. Here we report a multiphase NMR approach using magic‐angle spinning NMR to synchronously follow microbial processes of fermentation, lipid metabolism and structural dynamic changes in live microalgae cells. Chlamydomonas reinhardtii green algae were highly concentrated, introducing dark fermentation and anoxia conditions. Single‐pulse NMR experiments were applied to obtain temperature‐dependent kinetic profiles of the formed fermentation products. Through dynamics‐based spectral editing NMR, simultaneous conversion of galactolipids into TAG and free fatty acids was observed and rapid loss of rigid lipid structures. This suggests that lipolysis under dark and anoxia conditions finally results in the breakdown of cell and organelle membranes, which could be beneficial for recovery of intracellular microbial useful products. [ABSTRACT FROM AUTHOR]

Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magicangle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments--namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive twodimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ~6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems. [ABSTRACT FROM AUTHOR]

Alpha-synuclein (α-syn) is a small presynaptic protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). It localizes to presynaptic terminals where it partitions between a cytosolic soluble and a lipid-bound state. Recent evidence suggests that α-syn can also associate with mitochondrial membranes where it interacts with a unique anionic phospholipid cardiolipin (CL). Here, we examine the conformation of the flexible fragments of a monomeric α-syn bound to lipid vesicles composed of anionic 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, of tetraoleoyl CL (TOCL) and DOPC, and of fibrils. The dynamic properties of α-syn associated with DOPA:DOPC vesicles were the most favorable for conducting three-dimensional NMR experiments, and the 13C, 15N and amide 1H chemical shifts of the flexible and disordered C-terminus of α-syn could be assigned using three-dimensional through-bond magic angle spinning NMR spectroscopy. Although the C-terminus is more dynamically constrained in fibrils and in α-syn bound to TOCL:DOPC vesicles, a direct comparison of carbon chemical shifts detected using through bond two-dimensional spectroscopy indicates that the C-terminus is flexible and unstructured in all the three samples. [ABSTRACT FROM AUTHOR]

Molecular structure determination is the basis for understanding chemical processes and the property of materials. The direct dependence of the magnetic dipolar interaction on the distance makes solid‐state nuclear magnetic resonance (NMR) an excellent tool to study molecular structure when X‐ray crystallography fails to provide atomic‐resolution data. Although techniques to measure distances between pairs of isolated nuclear spin‐1/2 pairs are routine and easy to implement using the rotational echo double resonance (REDOR) experiment (Gullion & Schaefer, 1989), the existence of a nucleus with a spin > 1/2, appearing in approximately 75% of the elements in the periodic table, poses a challenge due to difficulties stemming from the large nuclear quadrupolar coupling constant (QCC). This mini‐review presents the existing solid‐state magic‐angle spinning NMR techniques aimed toward the efficient and accurate determination of internuclear distances between a spin‐1/2 and a "quadrupolar" nucleus having a spin larger than one half. Analytical expressions are provided for the various recoupling curves stemming from different techniques, and a coherent nomenclature for these various techniques is suggested. Treatment of some special cases such as multiple spin effects and spins with close Larmor frequencies is also discussed. The most advanced methods can recouple spins with quadrupolar frequencies up to tens of megahertz and beyond, expanding the distance measurement capabilities of solid‐state NMR to an increasingly growing number of applications and nuclear spin systems. [ABSTRACT FROM AUTHOR]

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 Å and a tilt of 36.1-36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances. [ABSTRACT FROM AUTHOR]

Deuterium rotating frame solid-state NMR relaxation measurements (2H R 1 ρ ) are important tools in quantitative studies of molecular dynamics. We demonstrate how 2H to 13C cross-polarization (CP) approaches under 10–40 kHz magic angle spinning rates can be combined with the 2H R 1 ρ blocks to allow for extension of deuterium rotating frame relaxation studies to methyl groups in biomolecules. This extension permits detection on the 13C nuclei and, hence, for the achievement of site-specific resolution. The measurements are demonstrated using a nine-residue low complexity peptide with the sequence GGKGMGFGL, in which a single selective −13CD 3 label is placed at the methionine residue. Carbon-detected measurements are compared with the deuterium direct-detection results, which allows for fine-tuning of experimental approaches. In particular, we show how the adiabatic respiration CP scheme and the double adiabatic sweep on the 2H and 13C channels can be combined with the 2H R 1 ρ relaxation rates measurement. Off-resonance 2H R 1 ρ measurements are investigated in addition to the on-resonance condition, as they extent the range of effective spin-locking field. [Display omitted] • Focus on 2H rotating frame relaxation in biomolecular solids. • Shown how 2H –13C CP can be combined with the rotating frame relaxation measurements. • Used synthetic nine residue disorder peptide with -13CD 3 label. [ABSTRACT FROM AUTHOR]

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144) has been linked to a heritable prionopathy in humans and is also capable of triggering a transmissible prion disease in mice. PrP23-144 can be converted from soluble monomeric form to amyloid under physiological conditions, providing an in vitro model for investigating the molecular basis of amyloid strains and cross-seeding barriers. Here, we use magic-angle spinning solid-state NMR to establish the sequential backbone and sidechain 13C and 15N chemical shift assignments for amyloid fibrils formed by the A117V and M129V mutants of human PrP23-144, which in the context of full length PrP in vivo are among the specific residues associated with development of Gerstmann–Straüssler–Scheinker disease. The chemical shift data are utilized to identify amino acids comprising the rigid amyloid core regions and to predict the protein secondary structures for human PrP23-144 A117V and M129V fibrils. [ABSTRACT FROM AUTHOR]

An important step in the process of protein research by NMR is the assignment of chemical shifts. In the coat protein of IKe bacteriophage, there are 53 residues making up a long helix resulting in relatively high spectral ambiguity. Assignment thus requires the collection of a set of three‐dimensional (3D) experiments and the preparation of sparsely labeled samples. Increasing the dimensionality can facilitate fast and reliable assignment of IKe and of larger proteins. Recent progress in nonuniform sampling techniques made the application of multidimensional NMR solid‐state experiments beyond 3D more practical. 4D 1H‐detected experiments have been demonstrated in high‐fields and at spinning speeds of 60 kHz and higher but are not practical at spinning speeds of 10–20 kHz for fully protonated proteins. Here, we demonstrate the applicability of a nonuniformly sampled 4D 13C/15N‐only correlation experiment performed at a moderate field of 14.1 T, which can incorporate sufficiently long acquisition periods in all dimensions. We show how a single CANCOCX experiment, supported by several 2D carbon‐based correlation experiments, is utilized for the assignment of heteronuclei in the coat protein of the IKe bacteriophage. One sparsely labeled sample was used to validate sidechain assignment of several hydrophobic‐residue sidechains. A comparison to solution NMR studies of isolated IKe coat proteins embedded in micelles points to key residues involved in structural rearrangement of the capsid upon assembly of the virus. The benefits of 4D to a quicker assignment are discussed, and the method may prove useful for studying proteins at relatively low fields. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the β-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol. [ABSTRACT FROM AUTHOR]

We report solid‐state 87Rb NMR spectra from two Rb‐ionophore complexes obtained with fast magic‐angle spinning (MAS) (up to 60 kHz) at 21.1 T. These Rb‐ionophore complexes containing macrocycles such as benzo‐15‐crown‐5 and cryptand [2.2.2] are typical of organic Rb salts that exhibit very large 87Rb quadrupole coupling constants (close to 20 MHz). We have also obtained static 87Rb NMR spectra for these two compounds and determined both 87Rb quadrupole coupling and chemical shift tensors. The experimental 87Rb NMR tensor parameters are compared with those obtained by quantum chemical computations. Our results demonstrate that the combination of fast MAS (60 kHz or higher) and a high magnetic field (21.1 T or higher) is sufficient to produce high‐quality solid‐state 87Rb NMR spectra for organic Rb solids at the natural abundance level. We anticipate that, with additional 87Rb isotope enrichment (up to 99%), the sensitivity of solid‐state 87Rb NMR will be 400 times higher than 39K NMR, which makes the former an attractive surrogate probe for studying K+ ion binding in biological systems. [ABSTRACT FROM AUTHOR]

The ¹H → 31P cross-polarization (CP) kinetics in the nanostructured calcium hydroxyapatite (nano-CaHA) was measured under moderate (5 kHz) magic-angle spinning (MAS) rate. This material was chosen as it contains the distanced 1H-31P spin pairs and the interactions between them are characterized by a relatively low dipolar coupling (b) that could be comparable with the spin-diffusion rates (R). Therefore, the physical legitimacy to use the secular solution of the quantum Liouville-von Neumann equation is doubtful. The semi-nonsecular model of spin dynamics was applied, and the results were compared with those obtained by the secular approach. The comparable results obtained by both models show that the secular model is applicable, with certain reservation, also in the case of |b| = R. The extremely high anisotropy of spin diffusion in the nano-CaHA was deduced. This can be a matter of the applied approach, as the interactions of the 31P spins with the proton bath were neglected in both models. The high anisotropy could also be caused by the physical reasons that stem from the structural and proton diffusion features of CaHA. This material belongs to low-dimensional proton conductors possessing a large motional freedom for protons along OH- chains. [ABSTRACT FROM AUTHOR]

The envelope (E) proteins of coronaviruses (CoVs) form cation-conducting channels that are associated with the pathogenicity of these viruses. To date, high-resolution structural information about these viroporins is limited to the SARS-CoV E protein. To broaden our structural knowledge of other members of this family of viroporins, we now investigate the conformation of the E protein of the human coronavirus (hCoV), NL63. Using two- and three-dimensional magic-angle-spinning NMR, we have measured 13 C and 15 N chemical shifts of the transmembrane domain of E (ETM), which yielded backbone (ϕ, ψ) torsion angles. We further measured the water accessibility of NL63 ETM at neutral pH versus acidic pH in the presence of Ca 2+ ions. These data show that NL63 ETM adopts a regular α-helical conformation that is unaffected by pH and the N-terminal ectodomain. Interestingly, the water accessibility of NL63 ETM increases only modestly at acidic pH in the presence of Ca 2+ compared to neutral pH, in contrast to SARS ETM, which becomes much more hydrated at acidic pH. This difference suggests a structural basis for the weaker channel conductance of α-CoV compared to β-CoV E proteins. The weaker E channel activity may in turn contribute to the reduced virulence of hCoV-NL63 compared to SARS-CoV viruses., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy., Competing Interests: Competing interests statement:The authors declare no competing interest.

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 1H, 7Li, and 19F 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 7Li and 19F 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. [ABSTRACT FROM AUTHOR]

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops. [ABSTRACT FROM AUTHOR]

Solid‐state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X‐ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three‐dimensional (3D) spectra needs to be acquired. Band‐selective homo‐nuclear cross‐polarization (BSH‐CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13C–15N‐labeled proteins by BSH‐CP‐based 13C‐detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side‐chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time‐saving approach. [ABSTRACT FROM AUTHOR]

The Eu2+-doped strontium aluminate SrAl4O7 samples have shown the blue-green persistent luminescence at 490 nm while the co-doping with Dy3+ shifts the maximum of emission to 475 nm. Undoped, 3% Eu-doped and 6% Dy-co-doped SrAl4O7 samples were prepared by the solid state-reaction method and studied by the solid-state 27Al MAS NMR applying the single pulse-acquire and Hahn-echo pulse sequences. It was shown that the Eu2+ with Dy3+ ion doping did not affect the bulk structure as well as the local Al environment in SrAl4O7. This means that large shifts of the emission maximum cannot be caused by changes in the local environment upon the co-doping of SrAl4O7:Eu2+ with Dy3+. However, the spectral features observed in the range between the signals of 4- and 6-coordinated Al (20-40 ppm) indicate that certain phase imperfections are present in all studied samples, and most probably amorphous/glassy domains were formed. Note that such amount of phase impurities was not detected by standard XRD or FTIR methods. This has revealed the 27Al MAS NMR technique to be a very effective tool monitoring the phase perfectness in series of strontium aluminate samples. [ABSTRACT FROM AUTHOR]

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Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1200 MHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

For the first time, a 1H solid-state photo-CIDNP effect is reported. The effect is observed in frozen solution of cyclohexanone in 1,4-dioxane-d8 under magic-angle spinning and continuous irradiation by the full spectrum of a xenon arc lamp. The spectral features show close similarity with those observed by 1H liquid-state photo-CIDNP NMR. [ABSTRACT FROM AUTHOR]

• A (S + 1/2) loss in many correlation and polarization-transfer experiments of quadrupole nuclei S. • Level-crossings of satellite transition and rf frequency under MAS complicate the spin dynamics of quadrupole nuclei. • NMR method development, high magnetic fields, efficient and high-power probes are important to quadrupole nuclei. High magnetic field can dramatically increase the spectral resolution and sensitivity of quadrupole nuclei S > 1/2 by the reduction of the second-order quadrupole broadening. A brief overview and outlook on spectral acquisition, the importance of high magnetic field, inter-nuclei distance measurement, various 2D separation and correlation methods of quadrupole nuclei are presented. The complications and consequences of spin dynamics under rf irradiation for the (2S + 1) level system and level-crossing with the satellite transition frequencies under magic-angle spinning are discussed. There is a scaling down of (S + 1/2) to the efficiency of many experiments in comparison with a spin-1/2 due to the fact that only two central transition spin states out of the (2S + 1) levels contribute to polarization transfer and spin correlation. [ABSTRACT FROM AUTHOR]

• Amyloids are filamentous supramolecular peptide or protein aggregates. • Amyloid formation is a hallmark of many neurodegenerative and other diseases. • Amyloid formation can be non-pathogenic and important for function. • Solid-state NMR provides atomic resolution data on amyloid structure and dynamics. In this perspective article I briefly highlight the rapid progress made over the past two decades in atomic level structural and dynamic studies of amyloids, which are representative of non-crystalline biomacromolecular assemblies, by magic-angle spinning solid-state NMR spectroscopy. Given new and continuing developments in solid-state NMR instrumentation and methodology, ongoing research in this area promises to contribute to an improved understanding of amyloid structure, polymorphism, interactions, assembly mechanisms, and biological function and toxicity. [ABSTRACT FROM AUTHOR]

• Magnetic field drift is nearly linear over short time periods of ∼12–36 h. • Linear data correction restores resolution and sensitivity. • Summation of short datasets and data correction can perform as a field lock. Magnetic field drift during magic angle spinning (MAS) NMR measurements is detrimental to the spectra, causing broadening of lines and distortion of lineshapes, especially in high-quality samples with linewidths of less than 0.1 ppm. We report that a simple linear correction for magnet drift can be used to improve the quality of proton detected MAS NMR measurements. Despite the fact that the magnetic field of superconducting magnets changes in a non-linear fashion, we find that when data acquisition is sufficiently short, a linear correction is a good approximation to the actual field drift. We used a script written in the C programming language for linear drift correction of multidimensional datasets (2D, 3D, 4D), which can be executed directly from Bruker Topspin. A second script allows datasets to be subdivided into arbitrarily short measurements, individually corrected, and concatenated before processing. [ABSTRACT FROM AUTHOR]

Abstract Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo 2H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies. Graphical abstract Unlabelled Image Highlights • Marine bacteria are studied by in vivo solid-state NMR for the first time. • 2H isotopic labelling of marine bacteria membranes is optimized. • Membrane fluidity measured by NMR changes with bacterial growth time. • Fatty acid analysis shows bacterial adaptation to labelling conditions. • Labelled bacteria harvested in the late exponential phase are in a native state. [ABSTRACT FROM AUTHOR]

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called "rotor crash" involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe ("CONFINE-MAS" probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid. [ABSTRACT FROM AUTHOR]

Nuclear Magnetic Resonance (NMR) has, over the past few decades, emerged as the most powerful spectroscopic technique for studying molecular structure across a sub-nanometer scale, as well as for probing molecular dynamics over widely spanning timescales (ns to s) [...]

Anabaena Sensory Rhodopsin (ASR) is a unique photochromic membrane-embedded photosensor which interacts with soluble transducer and is likely involved in a light-dependent gene regulation in the cyanobacterium Anabaena sp. PCC 7120. We report partial spectroscopic 1H, 13C and 15N assignments of perdeuterated and back-exchanged ASR reconstituted in lipids. The reported assignments are in general agreement with previously determined assignments of carbon and nitrogen resonances in fully protonated samples. Because the back-exchange was performed on ASR in a detergent-solubilized state, the location of detected residues reports on the solvent accessibility of ASR in detergent. A comparison with the results of previously published hydrogen/exchange data collected on the ASR reconstituted in lipids, suggests that the protein has larger solvent accessible surface in the detergent-solubilized state. [ABSTRACT FROM AUTHOR]

Fast magic-angle spinning, coupled with 1 H detection is a powerful method to improve spectral resolution and signal to noise in solid-state NMR spectra. Commercial probes now provide spinning frequencies in excess of 100 kHz. Then, one has sufficient resolution in the 1 H dimension to directly detect protons, which have a gyromagnetic ratio approximately four times larger than 13 C spins. However, the gains in sensitivity can quickly be lost if the rotation angle is not set precisely. The most common method of magic-angle calibration is to optimize the number of rotary echoes, or sideband intensity, observed on a sample of KBr. However, this typically uses relatively low spinning frequencies, where the spinning of fast-MAS probes is often unstable, and detection on the 13 C channel, for which fast-MAS probes are typically not optimized. Therefore, we compare the KBr-based optimization of the magic angle with two alternative approaches: optimization of the splitting observed in 13 C-labeled glycine-ethylester on the carbonyl due to the Cα–C′ J -coupling, or optimization of the H–N J -coupling spin echo in the protein sample itself. The latter method has the particular advantage that no separate sample is necessary for the magic-angle optimization. [ABSTRACT FROM AUTHOR]

Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural “hotspots” for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin. [ABSTRACT FROM AUTHOR]

Abstract: The capsid dynamics of filamentous bacteriophages is related to their function, stability, and interactions with the genome, and can be assessed by measuring the chemical shift anisotropy (CSA) of 15N amides, which are sensitive to large amplitude motions. In this study, CSA recoupling experiments under magic‐angle spinning NMR were used to probe the dynamics of the y21m capsid mutant of fd bacteriophage. Based on fitting the generated CSA lineshapes, residues located in the N‐terminus undergo increased motional amplitudes suggesting its global motion, whereas other backbone residues are rigid, and imply a tight hydrophobic packing of the phage. [ABSTRACT FROM AUTHOR]