17O Solid-State NMR Spectroscopy of Lipid Membranes.

Despite the limitations posed by poor sensitivity, studies have reported the unique advantages of 17O based NMR spectroscopy to study systems existing in liquid, solid, or semisolid states. 17O NMR studies have exploited the remarkable sensitivity of quadrupole coupling and chemical shift anisotropy tensors to the local environment in the characterization of a variety of intra- and intermolecular interactions and motion. Recent studies have considerably expanded the use of 17O NMR to study dynamic intermolecular interactions associated with some of the challenging biological systems under magic angle spinning (MAS) and aligned conditions. The very fast relaxing nature of 17O has been well utilized in cellular and in vivo MRS (magnetic resonance spectroscopy) and MRI (magnetic resonance imaging) applications. The main focus of this Review is to highlight the new developments in the biological solids with a detailed discussion for a few selected examples including membrane proteins and nanodiscs. In addition to the unique benefits and limitations, the remaining challenges to overcome, and the impacts of higher magnetic fields and sensitivity enhancement techniques are discussed.

Carbon-detected deuterium solid-state NMR rotating frame relaxation measurements for protein methyl groups under magic angle spinning.

Deuterium rotating frame solid-state NMR relaxation measurements (2H R1ρ) 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 R1ρ 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 -13CD3 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 R1ρ relaxation rates measurement. Off-resonance 2H R1ρ measurements are investigated in addition to the on-resonance condition, as they extent the range of effective spin-locking field.

Persistence of Methionine Side Chain Mobility at Low Temperatures in a Nine-Residue Low Complexity Peptide, as Probed by 2 H Solid-State NMR.

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-ß1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.

Host Defense Peptide Piscidin and Yeast-Derived Glycolipid Exhibit Synergistic Antimicrobial Action through Concerted Interactions with Membranes.

Developing new antimicrobials as alternatives to conventional antibiotics has become an urgent race to eradicate drug-resistant bacteria and to save human lives. Conventionally, antimicrobial molecules are studied independently even though they can be cosecreted in vivo. In this research, we investigate two classes of naturally derived antimicrobials: sophorolipid (SL) esters as modified yeast-derived glycolipid biosurfactants that feature high biocompatibility and low production cost; piscidins, which are host defense peptides (HDPs) from fish. While HDPs such as piscidins target the membrane of pathogens, and thus result in low incidence of resistance, SLs are not well understood on a mechanistic level. Here, we demonstrate that combining SL-hexyl ester (SL-HE) with subinhibitory concentration of piscidins 1 (P1) and 3 (P3) stimulates strong antimicrobial synergy, potentiating a promising therapeutic window. Permeabilization assays and biophysical studies employing circular dichroism, NMR, mass spectrometry, and X-ray diffraction are performed to investigate the mechanism underlying this powerful synergy. We reveal four key mechanistic features underlying the synergistic action: (1) P1/3 binds to SL-HE aggregates, becoming α-helical; (2) piscidin-glycolipid assemblies synergistically accumulate on membranes; (3) SL-HE used alone or bound to P1/3 associates with phospholipid bilayers where it induces defects; (4) piscidin-glycolipid complexes disrupt the bilayer structure more dramatically and differently than either compound alone, with phase separation occurring when both agents are present. Overall, dramatic enhancement in antimicrobial activity is associated with the use of two membrane-active agents, with the glycolipid playing the roles of prefolding the peptide, coordinating the delivery of both agents to bacterial surfaces, recruiting the peptide to the pathogenic membranes, and supporting membrane disruption by the peptide. Given that SLs are ubiquitously and safely used in consumer products, the SL/peptide formulation engineered and mechanistically characterized in this study could represent fertile ground to develop novel synergistic agents against drug-resistant bacteria.

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein.

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. At debate is its oligomeric state, let alone its function. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein was previously found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identify only a front-to-front, symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

On-Pathway Oligomer of Human Islet Amyloid Polypeptide Induced and Stabilized by Mechanical Rotation during Magic Angle Spinning Nuclear Magnetic Resonance.

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability under standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human type-2 diabetes-associated islet amyloid polypeptide (hIAPP or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant ß-sheet content and the capability of seeding hIAPP fibrillation. We used MAS nuclear magnetic resonance to demonstrate that the nature of the species was sensitive to sample conditions, including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

On-Pathway Oligomer of Human Islet Amyloid Polypeptide Induced and Stabilized by Mechanical Rotation During MAS NMR.

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant beta-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

The applications of solid-state NMR and MRI techniques in the study of rechargeable sodium-ion batteries.

In order to develop new electrode and electrolyte materials for advanced sodium-ion batteries (SIBs), it is crucial to understand a number of fundamental issues. These include the compositions of the bulk and interface, the structures of the materials used, and the electrochemical reactions in the batteries. Solid-state NMR (SS-NMR) has unique advantages in characterizing the local or microstructure of solid electrode/electrolyte materials and their interfaces-one such advantage is that these are determined in a noninvasive and nondestructive manner at the atomic level. In this review, we provide a survey of the recent advances in the understanding of the fundamental issues of SIBs using advanced NMR techniques. First, we summarize the applications of SS-NMR in characterizing electrode material structures and solid electrolyte interfaces (SEI). In particular, we elucidate the key role of in-situ NMR/MRI in revealing the complex reactions and degradation mechanisms of SIBs. Next, the characteristics and shortcomings of SS-NMR and MRI techniques in SIBs are also discussed in comparison to similar Li-ion batteries. Finally, an overview of SS-NMR and MRI techniques for sodium batteries are briefly discussed and presented.

Deuteron off-resonance rotating frame relaxation for the characterization of slow motions in rotating and static solid-state proteins.

We demonstrate the feasibility of deuterium solid-state NMR off-resonance rotating frame relaxation measurements for studies of slow motions in biomolecular solids. The pulse sequence, which includes adiabatic pulses for magnetization alignment, is illustrated for static and magic-angle spinning conditions away from rotary resonances. We apply the measurements for three systems with selective deuterium labels at methyl groups: a) a model compound, Fluorenylmethyloxycarbonyl methionine-D3 amino acid, for which the principles of the measurements and corresponding motional modeling based on rotameric interconversions are demonstrated; b) amyloid-ß1-40 fibrils labeled at a single alanine methyl group located in the disordered N-terminal domain. This system has been extensively studied in prior work and here serves as a test of the method for complex biological systems. The essential features of the dynamics consist of large-scale rearrangements of the disordered N-terminal domain and the conformational exchange between the free and bound forms of the domain, the latter one due to transient interactions with the structured core of the fibrils. and c) a 15-residue helical peptide which belongs to the predicted α-helical domain near the N-terminus of apolipoprotein B. The peptide is solvated with triolein and incorporates a selectively labeled leucine methyl groups. The method permits model refinement, indicating rotameric interconversions with a distribution of rate constants.

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein.

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

Orexin A, an amphipathic α-helical neuropeptide involved in pleiotropic functions in the nervous and immune systems: Synthetic approach and biophysical studies of the membrane-bound state.

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

Understanding the failure process of sulfide-based all-solid-state lithium batteries via operando nuclear magnetic resonance spectroscopy.

The performance of all-solid-state lithium metal batteries (SSLMBs) is affected by the presence of electrochemically inactive (i.e., electronically and/or ionically disconnected) lithium metal and solid electrolyte interphase (SEI), which are jointly termed inactive lithium. However, the differentiation and quantification of inactive lithium during cycling are challenging, and their lack limits the fundamental understanding of SSLMBs failure mechanisms. To shed some light on these crucial aspects, here, we propose operando nuclear magnetic resonance (NMR) spectroscopy measurements for real-time quantification and evolution-tracking of inactive lithium formed in SSLMBs. In particular, we examine four different sulfide-based solid electrolytes, namely, Li10GeP2S12, Li9.54Si1.74P1.44S11.7Cl0.3, Li6PS5Cl and Li7P3S11. We found that the chemistry of the solid electrolyte influences the activity of lithium. Furthermore, we demonstrate that electronically disconnected lithium metal is mainly found in the interior of solid electrolytes, and ionically disconnected lithium metal is found at the negative electrode surface. Moreover, by monitoring the Li NMR signal during cell calendar ageing, we prove the faster corrosion rate of mossy/dendritic lithium than flat/homogeneous lithium in SSLMBs.

Magnetically aligned nanodiscs enable direct measurement of 17O residual quadrupolar coupling for small molecules.

The use of 17O in NMR spectroscopy for structural studies has been limited due to its low natural abundance, low gyromagnetic ratio, and quadrupolar relaxation. Previous solution 17O work has primarily focused on studies of liquids where the 17O quadrupolar coupling is averaged to zero by isotropic molecular tumbling, and therefore has ignored the structural information contained in this parameter. Here, we use magnetically aligned polymer nanodiscs as an alignment medium to measure residual quadrupolar couplings (RQCs) for 17O-labelled benzoic acid in the aqueous phase. We show that increasing the magnetic field strength improves spectral sensitivity and resolution and that each satellite peak of the expected pentet pattern resolves clearly at 18.8 T. We observed no significant dependence of the RQC magnitudes on the magnetic field strength. However, changing the orientation of the alignment medium alters the RQC by a consistent factor, suggesting that 17O RQCs measured in this way can provide reliable orientational information for elucidations of molecular structures.

Dynamics of base pairs with low stability in RNA by solid-state nuclear magnetic resonance exchange spectroscopy.

Base pairs are fundamental building blocks of RNA. The base pairs of low stability are often critical in RNA functions. Here, we develop a solid-state NMR-based water-RNA exchange spectroscopy (WaterREXSY) to characterize RNA in solid. The approach uses different chemical exchange rates between iminos and water to evaluate base pair stability; the less stable ones would exchange more frequently, leading to stronger cross-peaks on WaterREXSY. Applied to the riboA71-adenine complex (the 71nt-aptamer domain of add adenine riboswitch from Vibrio vulnificus), the U47⋅U51 base pair, which is critical in ligand binding, was found to be less stable than other base pairs. The imino-water exchange rates of U47 at different temperatures are about 500-800 s-1, indeed indicative of low stability. This implies a highly complex and plastic triad involving U47⋅U51 and that the opening of the U47⋅U51 base pair may be the early stage of ligand release.

A machine learning protocol for revealing ion transport mechanisms from dynamic NMR shifts in paramagnetic battery materials.

Solid-state nuclear magnetic resonance (ssNMR) provides local environments and dynamic fingerprints of alkali ions in paramagnetic battery materials. Linking the local ionic environments and NMR signals requires expensive first-principles computational tools that have been developed for over a decade. Nevertheless, the assignment of the dynamic NMR spectra of high-rate battery materials is still challenging because the local structures and dynamic information of alkali ions are highly correlated and difficult to acquire. Herein, we develop a novel machine learning (ML) protocol that could not only quickly sample atomic configurations but also predict chemical shifts efficiently, which enables us to calculate dynamic NMR shifts with the accuracy of density functional theory (DFT). Using structurally well-defined P2-type Na2/3(Mg1/3Mn2/3)O2 as an example, we validate the ML protocol and show the significance of dynamic effects on chemical shifts. Moreover, with the protocol, it is demonstrated that the two experimental 23Na shifts (1406 and 1493 ppm) of P2-type Na2/3(Ni1/3Mn2/3)O2 originate from two stacking sequences of transition metal (TM) layers for the first time, which correspond to space groups P63/mcm and P6322, respectively. This ML protocol could help to correlate dynamic ssNMR spectra with the local structures and fast transport of alkali ions and is expected to be applicable to a wide range of fast dynamic systems.

Surprising Rigidity of Functionally Important Water Molecules Buried in the Lipid Headgroup Region.

Understanding water dynamics and structure is an important topic in biological systems. It is generally held in the literature that the interfacial water of hydrated phospholipids is highly mobile, in fast exchange with the bulk water ranging from the nano- to femtosecond timescale. Although nuclear magnetic resonance (NMR) is a powerful tool for structural and dynamic studies, direct probing of interfacial water in hydrated phospholipids is formidably challenging due to the extreme population difference between bulk and interfacial water. We developed a novel 17O solid-state NMR technique in combination with an ultra-high-field magnet (35.2 T) to directly probe the functionally important interfacial water. By selectively suppressing the dominant bulk water signal, we observed two distinct water species in the headgroup region of hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayers for the first time. One water species denoted as "confined water" is chemically and dynamically different from the bulk water (∼0.17 ppm downfield and a slightly shorter spin-lattice relaxation time). Another water species denoted as "bound water" has severely restricted motion and a distinct chemical shift (∼12 ppm upfield). Additionally, the bulk water is not as "free" as pure water, resulting from the fast exchange with the water molecules that weakly and transiently interact with the lipid choline groups. These new discoveries clearly indicate the existence of the interfacial water molecules that are relatively stable over the NMR timescale (on the order of milliseconds), providing an opportunity to characterize water dynamics on the millisecond or slower timescale in biomacromolecules.

Deuteron rotating frame relaxation for the detection of slow motions in rotating solids.

We demonstrate experimental and computational approaches for measuring 2H rotating frame NMR relaxation for solid samples under magic angle spinning (MAS) conditions. The relaxation properties of the deuterium spin-1 system are dominated by the reorientation of the anisotropic quadrupolar tensors, with the effective quadrupolar coupling constant around 55 kHz for methyl groups. The technique is demonstrated using the model compound dimethyl-sulfone at MAS rates of 10 and 60 kHz as well as for an amyloid fibril sample comprising an amyloid-ß (1-40) protein with a selective methyl group labeled in the disordered domain of the fibrils, at an MAS rate of 8 kHz. For both systems, the motional parameters fall well within the ranges determined by other techniques, thus validating its feasibility. Experimental and computational factors such as i) the probe's radio frequency inhomogeneity profiles, ii) rotary resonances at conditions for which the spin-lock field strength matches the half- or full-integer of the MAS rate, iii) the choice of MAS rates and spin-lock field strengths, and iv) simulations that account for the interconversion of multiple coherences for the spin-1 system under MAS and deviations from the analytical Redfield treatment are thoroughly considered.

Quantitatively analyzing the failure processes of rechargeable Li metal batteries.

Practical use of lithium (Li) metal for high­energy density lithium metal batteries has been prevented by the continuous formation of Li dendrites, electrochemically isolated Li metal, and the irreversible formation of solid electrolyte interphases (SEIs). Differentiating and quantifying these inactive Li species are key to understand the failure mode. Here, using operando nuclear magnetic resonance (NMR) spectroscopy together with ex situ titration gas chromatography (TGC) and mass spectrometry titration (MST) techniques, we established a solid foundation for quantifying the evolution of dead Li metal and SEI separately. The existence of LiH is identified, which causes deviation in the quantification results of dead Li metal obtained by these three techniques. The formation of inactive Li under various operating conditions has been studied quantitatively, which revealed a general "two-stage" failure process for the Li metal. The combined techniques presented here establish a benchmark to unravel the complex failure mechanism of Li metal.

Gas-Phase Fluorination of Hexagonal Boron Nitride.

Hexagonal boron nitride (hBN) has received much attention in recent years as a 2D dielectric material with potential applications ranging from catalysts to electronics. hBN is a stable covalent compound with a planar hexagonal lattice and is relatively unreactive to most chemical environments, making the chemical functionalization of hBN challenging. Here, a simple, scalable strategy to fluorinate hBN using a direct gas-phase fluorination technique is reported. The nature of fluorine bonding to the hBN lattice and their chemical coordination are described based on various characterization studies and theoretical models. The fluorine functionalized hBN shows a bandgap reduction and displays a semiconducting behavior due to the fluorination process. Additionally, the fluorinated hBN shows significant improvement in its thermal and friction properties, which could be substantial in applications such as lubricants and thermal fluids. Theory and simulations reveal that the enhanced friction properties of fluorinated hBN result from reduced inter-planar interaction energy by electrostatic repulsion of intercalated fluorine atoms between hBN layers without significant disruption of the in-plane lattice. This technique paves the way for the fluorination of several other 2D structures for various applications such as magnetism and functional nanoscale electronic devices.

Triple-pulse excitation: An efficient way for suppressing background signals and eliminating radio-frequency acoustic ringing in direct polarization NMR experiments.

Direct polarization using a single pulse is the simplest excitation scheme in nuclear magnetic resonance (NMR) experiments, capable of quantifying various compositions in many materials applications. However, this single-pulse excitation generally gives rise to NMR spectra with a severely distorted baseline due to the background signals arising from probe components and/or due to the radio-frequency (RF) acoustic ringing, especially in low-γ nuclei and wide-line NMR. In this work, a triple-pulse excitation scheme is proposed to simultaneously suppress the background signals and eliminate the RF acoustic ringing. The acoustic ringing is cancelled through subtraction in any two consecutive scans by alternating the receiver phase while keeping the phase of the pulse right before acquisition the same. While the triple-pulse scheme generates an additional flip-angle dependent scaling to the traditional single-pulse excitation profile in such a way that the scaling is one when the flip-angle is ∼90° but becomes almost zero when the flip-angle is very small. Therefore, the background signals arising from the materials outside the sample coil experiencing a very small fraction of the RF flip-angles can be effectively suppressed. Various samples containing 1H and quadrupolar nuclei (17O, 25Mg, and 23Na) have been used to demonstrate the effectiveness of this newly proposed triple-pulse excitation in terms of suppressing the background signals and eliminating the acoustic ringing effects.