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The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to understanding membrane protein biology. We report Native-nanoBleach, a total internal reflection fluorescence microscopy-based single-molecule photobleaching step analysis technique to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ~10 nm. We achieved this by capturing target membrane proteins in native nanodiscs with their proximal native membrane environment using amphipathic copolymers. We applied Native-nanoBleach to quantify the oligomerization status of structurally and functionally diverse membrane proteins, including a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under growth-factor binding and oncogenic mutations, respectively. Our data suggest that Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes under physiologically and clinically relevant conditions. Native-nanoBleach, a single-molecule imaging technique with a spatial resolution of ~10 nm, quantifies the oligomeric distribution of membrane proteins directly from native membranes at endogenous expression levels with their proximal native membrane environment using amphipathic copolymer nanodiscs.

Membrane lipids such as cardiolipin act as molecular glue to preserve the oligomeric states of membrane proteins with low oligomeric stability. Membrane-protein stabilization by lipid binding It is well established that lipid binding leads to the oligomerization of membrane proteins, and hence the activation of many signalling pathways, but it is not clear how the lipid bilayer affects the structure and function of membrane–protein complexes. Carol Robinson and colleagues have developed a mass-spectroscopy-based technique that allows the observation of oligomeric membrane–protein complexes with sufficient resolution to characterize their bound lipids. Using this technique, they evaluate the strength of oligomer formation for some 125 α-helical membrane proteins, including G-protein-coupled receptors. They find that lipid binding modulates protein interfaces, perturbing their monomer–oligomer equilibria, and show that manipulation of lipid binding can modify oligomer stability. And they use modelling to investigate possible binding sites for lipid molecules at the interfaces involved in oligomerization. These findings could aid the optimization of membrane–protein complexes for structural analysis. Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways 1 but is often difficult to define 2 or predict 3 . Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT 4 , one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET 5 , another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na + /H + antiporter NhaA from E. coli , which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

A high-resolution, Orbitrap-based, native mass spectrometry approach allows the direct characterization of lipid, peptide and drug binding to intact membrane proteins. Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry–based method for analyzing intact membrane protein–ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.

Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create a negative electrostatic surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases. Xkr4 apoptotic scramblase activation is thought to involve caspase cleavage and dimerization to expose phosphatidylserine on dying cells. The authors show that full-length Xkr4 is an active monomeric scramblase. CryoEM reveals a conformation that promotes membrane thinning and scrambling, enhancing understanding of hXkr4 activity.

Na + /H + antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na + /H + exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane. Na + /H + antiporters transport sodium or lithium ions across the membrane in exchange for protons. Here the authors combine ion mobility mass spectrometry and molecular dynamics simulations to uncover a facilitating role for lipids in the transport mechanism.

Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D -aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii . By comparing the biophysical properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability. Succinimide is a post-translational modification susceptible to rapid hydrolysis and generally associated with protein destabilisation. Here, the authors use mass spectroscopy to identify a stable succinimide intermediate that is responsible for the high thermostability of a thermophilic enzyme.

Mass spectrometric analysis of a banyan endophyte, Bacillus subtilis K1, extract showing broad spectrum antifungal activity revealed a complex mixture of lipopeptides, iturins, surfactins, and fengycins. Fractionation by reversed-phase high performance liquid chromatography (HPLC) facilitated a detailed analysis of fengycin microheterogeneity. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric studies permitted the identification of several new fengycin variants. Four major sites of heterogeneity are identified: (1) N-terminus β-hydroxy fatty acid moiety, where chain length variation and the presence of unsaturation occur, (2) position 6 (Ala/Val/Ile/Leu), (3) position 10 (Val/Ile) within the macrocyclic ring, and (4) Gln to Glu replacement at position 8, resulting in fengycin variants that differ in mass by 1 Da. Diagnostic fragment ions provide a quick method for localizing the sites of variation in the macrocycle or the linear segment. Subsequent establishment of the sequences is achieved by MS/MS analysis of linear fengycin species produced by hydrolysis of the macrocyclic lactone. Unsaturation in the fatty acid chain and the presence of linear precursors in the B. subtilis K1 extract are also established by mass spectrometry. The anomalous distribution of intensities within isotopic multiplets is a diagnostic for Gln/Glu replacements. High resolution mass spectrometry facilitates the identification of fengycin species differing by 1 Da by localizing the variable position (Gln 8 /Glu 8 ) in the fengycin variants.

This work presents optimized experimental protocols for the extraction of >2,000 unique membrane proteins directly from their native membranes into native nanodiscs. Combined with orthogonal structural, biochemical or biophysical approaches, these native nanodiscs enable membrane proteins to be studied directly in their native membrane context.

OmcZ nanowires produced by Geobacter species have high electron conductivity (>30 S cm −1 ). Of 111 cytochromes present in G. sulfurreducens , OmcZ is the only known nanowire-forming cytochrome essential for the formation of high-current-density biofilms that require long-distance (>10 µm) extracellular electron transport. However, the mechanisms underlying OmcZ nanowire assembly and high conductivity are unknown. Here we report a 3.5-Å-resolution cryogenic electron microscopy structure for OmcZ nanowires. Our structure reveals linear and closely stacked haems that may account for conductivity. Surface-exposed haems and charge interactions explain how OmcZ nanowires bind to diverse extracellular electron acceptors and how organization of nanowire network re-arranges in different biochemical environments. In vitro studies explain how G. sulfurreducens employ a serine protease to control the assembly of OmcZ monomers into nanowires. We find that both OmcZ and serine protease are widespread in environmentally important bacteria and archaea, thus establishing a prevalence of nanowire biogenesis across diverse species and environments. Cryo-EM structure of Geobacter cytochrome OmcZ nanowires identifies mechanism of electron conductance.

The native membrane environment profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergents to disrupt and remove this vital membrane context, impeding our ability to decipher the local molecular context and its effect. Here we develop a membrane proteome-wide platform that enables rapid spatially resolved extraction of target MPs directly from cellular membranes into native nanodiscs that maintain the local membrane context, using a library of membrane-active polymers. We accompany this with an open-access database that quantifies the polymer-specific extraction efficiency for 2,065 unique mammalian MPs and provides the most optimized extraction condition for each. To validate, we demonstrate how this resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, we show how the database can be extended to capture overexpressed multiprotein complexes by taking two synaptic vesicle MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane ‘nano-scoops’ containing a target MP and interface with structural, functional and bioanalytical approaches. This manuscript reports a high-throughput platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs by using a library of membrane-active polymers while maintaining their local membrane context.