17O NMR Spectroscopy in Lithium-Ion Battery Cathode Materials: Challenges and Interpretation.

Modern studies of lithium-ion battery (LIB) cathode materials employ a large range of experimental and theoretical techniques to understand the changes in bulk and local chemical and electronic structures during electrochemical cycling (charge and discharge). Despite its being rich in useful chemical information, few studies to date have used 17O NMR spectroscopy. Many LIB cathode materials contain paramagnetic ions, and their NMR spectra are dominated by hyperfine and quadrupolar interactions, giving rise to broad resonances with extensive spinning sideband manifolds. In principle, careful analysis of these spectra can reveal information about local structural distortions, magnetic exchange interactions, structural inhomogeneities (Li+ concentration gradients), and even the presence of redox-active O anions. In this Perspective, we examine the primary interactions governing 17O NMR spectroscopy of LIB cathodes and outline how 17O NMR may be used to elucidate the structure of pristine cathodes and their structural evolution on cycling, providing insight into the challenges in obtaining and interpreting the spectra. We also discuss the use of 17O NMR in the context of anionic redox and the role this technique may play in understanding the charge compensation mechanisms in high-capacity cathodes, and we provide suggestions for employing 17O NMR in future avenues of research.

Bulk fatigue induced by surface reconstruction in layered Ni-rich cathodes for Li-ion batteries.

Ni-rich layered cathode materials are among the most promising candidates for high-energy-density Li-ion batteries, yet their degradation mechanisms are still poorly understood. We report a structure-driven degradation mechanism for NMC811 (LiNi0.8Mn0.1Co0.1O2), in which a proportion of the material exhibits a lowered accessible state of charge at the end of charging after repetitive cycling and becomes fatigued. Operando synchrotron long-duration X-ray diffraction enabled by a laser-thinned coin cell shows the emergence and growth in the concentration of this fatigued phase with cycle number. This degradation is structure driven and is not solely due to kinetic limitations or intergranular cracking: no bulk phase transformations, no increase in Li/Ni antisite mixing and no notable changes in the local structure or Li-ion mobility of the bulk are seen in aged NMCs. Instead, we propose that this degradation stems from the high interfacial lattice strain between the reconstructed surface and the bulk layered structure that develops when the latter is at states of charge above a distinct threshold of approximately 75%. This mechanism is expected to be universal in Ni-rich layered cathodes. Our findings provide fundamental insights into strategies to help mitigate this degradation process.

Bulk and Surface Chemistry of the Niobium MAX and MXene Phases from Multinuclear Solid-State NMR Spectroscopy.

MXenes, derived from layered MAX phases, are a class of two-dimensional materials with emerging applications in energy storage, electronics, catalysis, and other fields due to their high surface areas, metallic conductivity, biocompatibility, and attractive optoelectronic properties. MXene properties are heavily influenced by their surface chemistry, but a detailed understanding of the surface functionalization is still lacking. Solid-state nuclear magnetic resonance (NMR) spectroscopy is sensitive to the interfacial chemistry, the phase purity including the presence of amorphous/nanocrystalline phases, and the electronic properties of the MXene and MAX phases. In this work, we systematically study the chemistry of Nb MAX and MXene phases, Nb2AlC, Nb4AlC3, Nb2CTx, and Nb4C3Tx, with their unique electronic and mechanical properties, using solid-state NMR spectroscopy to examine a variety of nuclei (1H, 13C, 19F, 27Al, and 93Nb) with a range of one- and two-dimensional correlation, wide-line, high-sensitivity, high-resolution, and/or relaxation-filtered experiments. Hydroxide and fluoride terminations are identified, found to be intimately mixed, and their chemical shifts are compared with other MXenes. This multinuclear NMR study demonstrates that diffraction alone is insufficient to characterize the phase composition of MAX and MXene samples as numerous amorphous or nanocrystalline phases are identified including NbC, AlO6 species, aluminum nitride or oxycarbide, AlF3·nH2O, Nb metal, and unreacted MAX phase. To the best of our knowledge, this is the first study to examine the transition-metal resonances directly in MXene samples, and the first 93Nb NMR of any MAX phase. The insights from this work are employed to enable the previously elusive assignment of the complex overlapping 47/49Ti NMR spectrum of Ti3AlC2. The results and methodology presented here provide fundamental insights on MAX and MXene phases and can be used to obtain a more complete picture of MAX and MXene chemistry, to prepare realistic structure models for computational screening, and to guide the analysis of property measurements.

Intrinsic Kinetic Limitations in Substituted Lithium-Layered Transition-Metal Oxide Electrodes.

Substituted Li-layered transition-metal oxide (LTMO) electrodes such as LixNiyMnzCo1-y-zO2 (NMC) and LixNiyCo1-y-zAlzO2 (NCA) show reduced first cycle Coulombic efficiency (90-87% under standard cycling conditions) in comparison with the archetypal LixCoO2 (LCO; ∼98% efficiency). Focusing on LixNi0.8Co0.15Al0.05O2 as a model compound, we use operando synchrotron X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) spectroscopy to demonstrate that the apparent first-cycle capacity loss is a kinetic effect linked to limited Li mobility at x > 0.88, with near full capacity recovered during a potentiostatic hold following the galvanostatic charge-discharge cycle. This kinetic capacity loss, unlike many capacity losses in LTMOs, is independent of the cutoff voltage during delithiation and it is a reversible process. The kinetic limitation manifests not only as the kinetic capacity loss during discharge but as a subtle bimodal compositional distribution early in charge and, also, a dramatic increase of the charge-discharge voltage hysteresis at x > 0.88. 7Li NMR measurements indicate that the kinetic limitation reflects limited Li transport at x > 0.86. Electrochemical measurements on a wider range of LTMOs including Lix(Ni,Fe)yCo1-yO2 suggest that 5% substitution is sufficient to induce the kinetic limitation and that the effect is not limited to Ni substitution. We outline how, in addition to a reduction in the number of Li vacancies and shrinkage of the Li-layer size, the intrinsic charge storage mechanism (two-phase vs solid-solution) and localization of charge give rise to additional kinetic barriers in NCA and nonmetallic LTMOs in general.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

G Protein-Coupled Receptors Contain Two Conserved Packing Clusters.

G protein-coupled receptors (GPCRs) have evolved a seven-transmembrane helix framework that is responsive to a wide range of extracellular signals. An analysis of the interior packing of family A GPCR crystal structures reveals two clusters of highly packed residues that facilitate tight transmembrane helix association. These clusters are centered on amino acid positions 2.47 and 4.53, which are highly conserved as alanine and serine, respectively. Ala2.47 mediates the interaction between helices H1 and H2, while Ser4.53 mediates the interaction between helices H3 and H4. The helical interfaces outside of these clusters are lined with residues that are more loosely packed, a structural feature that facilitates motion of helices H5, H6, and H7, which is required for receptor activation. Mutation of the conserved small side chain at position 4.53 within packing cluster 2 is shown to disrupt the structure of the visual receptor rhodopsin, whereas sites in packing cluster 1 (e.g., positions 1.46 and 2.47) are more tolerant to mutation but affect the overall stability of the protein. These findings reveal a common structural scaffold of GPCRs that is important for receptor folding and activation.

The Molecular Switching Mechanism at the Conserved D(E)RY Motif in Class-A GPCRs.

The disruption of ionic and H-bond interactions between the cytosolic ends of transmembrane helices TM3 and TM6 of class-A (rhodopsin-like) G protein-coupled receptors (GPCRs) is a hallmark for their activation by chemical or physical stimuli. In the bovine photoreceptor rhodopsin, this is accompanied by proton uptake at Glu(134) in the class-conserved D(E)RY motif. Studies on TM3 model peptides proposed a crucial role of the lipid bilayer in linking protonation to stabilization of an active state-like conformation. However, the molecular details of this linkage could not be resolved and have been addressed in this study by molecular dynamics (MD) simulations on TM3 model peptides in a bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We show that protonation of the conserved glutamic acid alters the peptide insertion depth in the membrane, its side-chain rotamer preferences, and stabilizes the C-terminal helical structure. These factors contribute to the rise of the side-chain pKa (> 6) and to reduced polarity around the TM3 C terminus as confirmed by fluorescence spectroscopy. Helix stabilization requires the protonated carboxyl group; unexpectedly, this stabilization could not be evoked with an amide in MD simulations. Additionally, time-resolved Fourier transform infrared (FTIR) spectroscopy of TM3 model peptides revealed a different kinetics for lipid ester carbonyl hydration, suggesting that the carboxyl is linked to more extended H-bond clusters than an amide. Remarkably, this was seen as well in DOPC-reconstituted Glu(134)- and Gln(134)-containing bovine opsin mutants and demonstrates that the D(E)RY motif is a hydrated microdomain. The function of the D(E)RY motif as a proton switch is suggested to be based on the reorganization of the H-bond network at the membrane interface.

Amino acid conservation and interactions in rhodopsin: probing receptor activation by NMR spectroscopy.

Rhodopsin is a classical two-state G protein-coupled receptor (GPCR). In the dark, its 11-cis retinal chromophore serves as an inverse agonist to lock the receptor in an inactive state. Retinal-protein and protein-protein interactions have evolved to reduce the basal activity of the receptor in order to achieve low dark noise in the visual system. In contrast, absorption of light triggers rapid isomerization of the retinal, which drives the conversion of the receptor to a fully active conformation. Several specific protein-protein interactions have evolved that maintain the lifetime of the active state in order to increase the sensitivity of this receptor for dim-light vision in vertebrates. In this article, we review the molecular interactions that stabilize rhodopsin in the dark-state and describe the use of solid-state NMR spectroscopy for probing the structural changes that occur upon light-activation. Amino acid conservation provides a guide for those interactions that are common in the class A GPCRs as well as those that are unique to the visual system. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Do plants contain g protein-coupled receptors?

Whether G protein-coupled receptors (GPCRs) exist in plants is a fundamental biological question. Interest in deorphanizing new GPCRs arises because of their importance in signaling. Within plants, this is controversial, as genome analysis has identified 56 putative GPCRs, including G protein-coupled receptor1 (GCR1), which is reportedly a remote homolog to class A, B, and E GPCRs. Of these, GCR2 is not a GPCR; more recently, it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix-alignment method, which has been benchmarked against the class A-class B-class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologs to class A, class B, and class F GPCRs and shown that GCR1 is closer to class A and/or class B GPCRs than class A, class B, or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the six GPCR classes. Variability comparisons provide additional evidence that GCR1 homologs have the GPCR fold. From the alignments and a GCR1 comparative model, we have identified motifs that are common to GCR1, class A, B, and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fold.

Retinitis pigmentosa mutants provide insight into the role of the N-terminal cap in rhodopsin folding, structure, and function.

Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.

Highly conserved tyrosine stabilizes the active state of rhodopsin.

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg135(3.50) and Met257(6.40) forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu134(3.49)-Arg135(3.50)-Glu247(6.30) ionic lock. We also show that Tyr223(5.58) and Tyr306(7.53) form molecular contacts with Met257(6.40). Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr223(5.58) and Ala132(3.47) in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr223(5.58) to phenylalanine or mutation of Ala132(3.47) to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr306(7.53) to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.

Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory.

Here I will review the development of gene expression systems for production of bovine rhodopsin in the Khorana laboratory with particular focus on stable mammalian cell lines made using human embryonic kidney cells (HEK293S). The synthesis of a gene encoding bovine rhodopsin was completed in 1986. This gene was expertly designed with the built-in capacity for DNA duplex cassette replacement mutagenesis which made site-directed mutagenesis relatively straightforward. Intense effort was expended over several years in order to identify a gene expression system capable of producing rhodopsin in milligram amounts as required for biophysical studies. Mammalian expression systems, both transient and stable, were found to be the most favourable based on several criteria including receptor expression levels, correct folding and post translational processing, and capacity for purification of fully functional receptor. Transient expression using COS-1 cells was preferred for routine small-scale production of rhodopsin mutants, while HEK293S stable cell lines were used when milligram amounts of rhodopsin mutants were needed; for example, when conducting NMR studies.

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the ß(2)-adrenergic receptor.

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the ß(2)-adrenergic receptor (ß(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing ß(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.

Construction of Recombinant Cell Lines for GPCR Expression.

Large-scale recombinant expression of G protein-coupled receptors (GPCRs) is required for structure and function studies where there is a need for milligram amounts of protein in pure form. Here we describe a procedure for the construction of human embryonic kidney 293S (HEK293S) stable cell lines for inducible expression of the gene encoding bovine rhodopsin. The HEK293S cell line is particularly suitable for this application because of several favorable properties as a recombinant host including: its ease of transfection, its capacity for handling large amounts of protein cargo, and its ability to perform the necessary co- and post-translational modifications required for correct folding and processing of complex membrane proteins such as GPCRs. The procedures described here will focus on the HEK293S GnTI- cell line, an HEK293S derivative that is widely used for the production of glycoproteins modified homogeneously with truncated N-glycans.

Structural Origins of Voltage Hysteresis in the Na-Ion Cathode P2-Na0.67[Mg0.28Mn0.72]O2: A Combined Spectroscopic and Density Functional Theory Study.

P2-layered sodium-ion battery (NIB) cathodes are a promising class of Na-ion electrode materials with high Na+ mobility and relatively high capacities. In this work, we report the structural changes that take place in P2-Na0.67[Mg0.28Mn0.72]O2. Using ex situ X-ray diffraction, Mn K-edge extended X-ray absorption fine structure, and 23Na NMR spectroscopy, we identify the bulk phase changes along the first electrochemical charge-discharge cycle-including the formation of a high-voltage "Z phase", an intergrowth of the OP4 and O2 phases. Our ab initio transition state searches reveal that reversible Mg2+ migration in the Z phase is both kinetically and thermodynamically favorable at high voltages. We propose that Mg2+ migration is a significant contributor to the observed voltage hysteresis in Na0.67[Mg0.28Mn0.72]O2 and identify qualitative changes in the Na+ ion mobility.

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N',N'-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

Closed loop folding units from structural alignments: experimental foldons revisited.

Nonoverlapping closed loops of around 25-35 amino acids formed via nonlocal interactions at the loop ends have been proposed as an important unit of protein structure. This hypothesis is significant as such short loops can fold quickly and so would not be bound by the Leventhal paradox, giving insight into the possible nature of the funnel in protein folding. Previously, these closed loops have been identified either by sequence analysis (conservation and autocorrelation) or studies of the geometry of individual proteins. Given the potential significance of the closed loop hypothesis, we have explored a new strategy for determining closed loops from the insertions identified by the structural alignment of proteins sharing the same overall fold. We determined the locations of the closed loops in 37 pairs of proteins and obtained excellent agreement with previously published closed loops. The relevance of NMR structures to closed loop determination is briefly discussed. For cytochrome c, cytochrome b(562) and triosephophate isomerase, independent folding units have been determined on the basis of hydrogen exchange experiments and misincorporation proton-alkyl exchange experiments. The correspondence between these experimentally derived foldons and the theoretically derived closed loops indicates that the closed loop hypothesis may provide a useful framework for analyzing such experimental data.

A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin.

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation.

Short-range ordering in a battery electrode, the 'cation-disordered' rocksalt Li1.25Nb0.25Mn0.5O2.

Cation order, with a local structure related to γ-LiFeO2, is observed in the nominally cation-disordered Li-excess rocksalt Li1.25Nb0.25Mn0.5O2via X-ray diffraction, neutron pair distribution function analysis, magnetic susceptibility and NMR spectroscopy. The correlation length of ordering depends on synthesis conditions and has implications for the electrochemistry of these phases.

The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.