Native Membrane Environment Research Articles

Functional roles of tyrosine 185 during the bacteriorhodopsin photocycle as revealed by in situ spectroscopic studies

Tyrosine 185 (Y185), one of the aromatic residues within the retinal (Ret) chromophore binding pocket in helix F of bacteriorhodopsin (bR), is highly conserved among the microbial rhodopsin family proteins. Many studies have investigated the functions of Y185, but its underlying mechanism during the bR photocycle remains unclear. To address this research gap, in situ two-dimensional (2D) magic-angle spinning (MAS) solid-state NMR (ssNMR) of specifically labelled bR, combined with light-induced transient absorption change measurements, dynamic light scattering (DLS) measurements, titration analysis and site-directed mutagenesis, was used to elucidate the functional roles of Y185 during the bR photocycle in the native membrane environment. Different interaction modes were identified between Y185 and the Ret chromophore in the dark-adapted (inactive) state and M (active) state, indicating that Y185 may serve as a rotamer switch maintaining the protein dynamics, and plays an important role in the efficient proton-pumping mechanism in the bR purple membrane.

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane.

Production and Structural Analysis of Membrane-Anchored Proteins in Phospholipid Nanodiscs.

Structural studies on membrane-anchored proteins containing a transmembrane (TM) helix have been hampered by difficulties in producing these proteins in a natively folded form. Detergents that are required to solubilize the hydrophobic TM helix usually destabilize the soluble domain. Thus, TM helices are removed for structural studies, which neglects the pivotal role of a membrane on protein function. This work presents a versatile strategy for the production of this protein class attached to phospholipid nanodiscs. By inserting the TM-helix into nanodiscs and a subsequent SortaseA-mediated ligation of the soluble domain, membrane-anchored BclxL could be obtained in a folded conformation. This strategy is suitable for high-resolution structure determination as well as for probing membrane location by NMR. This method will be applicable to a wide range of membrane-anchored proteins and will be useful to decipher their functional role in a native membrane environment.

Reconstitution of Voltage-Activated Potassium Channel into Phospholipid Bilayer

Voltage-activated ion channels are expressed in the membranes of excitable cells, and open and close in response to changes in membrane voltage. Physiologically, they play diverse roles, from the generation of action potentials to triggering the release of neurotransmitters at synapses. To study the biochemical properties, these ion channels are usually purified and studied under detergent-solubilized condition, but many ion channels behave differently in detergent when compared to their native membrane environment. One recently introduced technique to overcome this issue is using lipid bilayer nanodiscs. Nanodiscs are a few nanometer-sized hockey-puck-shaped lipid bilayers solubilized by engineered apolipoprotein, membrane scaffold protein (MSP). MSP is comprised of short repeats of amphipathic alpha helies that surround the hydrophobic acyl chains of phospholipids. To investigate the structure and biochemical properties of voltage-activated potassium (Kv) channels in a membrane environment, we reconstituted a Kv channel into phospholipid bilayer nanodiscs using phospholipid and MSP. We first attempted to assemble empty nandiscs (nanodisc without Kv) to determine the optimal ratio between lipid and MSP that provides the best yields. Using these conditions, we reconstituted Kv channels into nanodiscs, and show that they elute as a single peak in gel-filtration chromatography. SDS-PAGE result indicates that the preparation contains each subunit corresponding to the Kv and MSP, confirming successful reconstitution of the Kv into nanodiscs. We also show that a voltage-sensor toxin can bind to the Kv channel in nanodiscs, confirming that the architecture of the Kv channel is well maintained in nanodiscs.

Membrane protein insertion through a mitochondrial β-barrel gate.

The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of β-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of β-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of β-barrel proteins.

Making your way through the side of a barrel

Membrane Proteins The mechanism of membrane insertion and assembly of Β-barrel proteins is a central question of outer membrane biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria. Hohr et al. developed assays to address this fundamental problem. They systematically mapped precursor proteins transported by the mitochondrial Omp85 channel (Sam50) to elucidate the entire membrane insertion pathway of a precursor in the native membrane environment. Their findings directly demonstrate translocation of precursor proteins through the lumen of the mitochondrial Omp85 channel, signal recognition by β-strand exchange between channel and precursor, and exit through the lateral gate into the membrane. Science , this issue p. [eaah6834][1] [1]: /lookup/doi/10.1126/science.aah6834

A New Strain Collection for Improved Expression of Outer Membrane Proteins.

Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform

Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.

Morphologies of synaptic protein membrane fusion interfaces

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.

Multiplexed Lipid Bilayers on Silica Microspheres for Analytical Screening Applications.

Most druggable targets are membrane components, including membrane proteins and soluble proteins that interact with ligands or receptors embedded in membranes. Current target-based screening and intermolecular interaction assays generally do not include the lipid membrane environment in presenting these targets, possibly altering their native structure and leading to misleading or incorrect results. To address this issue, an ideal assay involving membrane components would (1) mimic the natural membrane environment, (2) be amenable to high-throughput implementation, and (3) be easily multiplexed. In a step toward developing such an ideal target-based analytical assay for membrane components, we present fluorescently indexed multiplexed biomimetic membrane assays amenable to high-throughput flow cytometric detection. We build fluorescently multiplexed biomimetic membrane assays by using varying amounts of a fluorescently labeled lipid, NBD-DOPE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)], incorporated into a phospholipid membrane bilayer supported on 3 μm silica microspheres. Using flow cytometry, we demonstrate this multiplexed approach by measuring specific affinity of two well-characterized systems, the fluorescently labeled soluble proteins cholera toxin B subunit-Alexa 647 and streptavidin-PE/Cy5, to membranes containing different amounts of ligand targets of these proteins, GM1 and biotin-DOPE, respectively. This work will enable future efforts in developing highly efficient biomimetic assays for interaction analysis and drug screening involving membrane components.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.

Oscillating Electric Field Measures the Rotation Rate in a Native Rotary Enzyme

Rotary enzymes are complex, highly challenging biomolecular machines whose biochemical working mechanism involves intersubunit rotation. The true intrinsic rate of rotation of any rotary enzyme is not known in a native, unmodified state. Here we use the effect of an oscillating electric (AC) field on the biochemical activity of a rotary enzyme, the vacuolar proton-ATPase (V-ATPase), to directly measure its mean rate of rotation in its native membrane environment, without any genetic, chemical or mechanical modification of the enzyme, for the first time. The results suggest that a transmembrane AC field is able to synchronise the steps of ion-pumping in individual enzymes via a hold-and-release mechanism, which opens up the possibility of biotechnological exploitation. Our approach is likely to work for other transmembrane ion-transporting assemblies, not only rotary enzymes, to determine intrinsic in situ rates of ion pumping.

Systematic protein–protein interaction mapping for clinically relevant human GPCRs

G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I

Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment.

Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells.

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2Kb , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2Kb and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.

In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain (TM) Structure and Dimerization in Native E. Coli Membranes

Cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases leads to generation of amyloid peptide (Aβ), which is the primary constituent of Alzheimer's disease (AD) plaques in the brain. Three consecutive GXXXG motifs and one GXXXA motif on the transmembrane domain (TM) of APP have been reported to be one or two important sites for mediating APP homodimerization in different experimental conditions, the difference are likely due to the membrane mimetic. We and several other groups recently demonstrated the feasibility to directly characterize recombinant proteins in native Escherichia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several strategies to further improve spectral sensitivity and resolution, and suppress background signals of E. coli proteins and lipids, including preparation of E. coli inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal filtering with the frequency-selective REDOR pulse sequence. These improvements have allowed us to characterize the structure and dimerization of APP TM and its variants in a native membrane environment.

Fusion Reporter Approaches to Monitoring Transmembrane Helix Interactions in Bacterial Membranes.

In transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but might also be mediated by helix-helix contacts in the inner membrane. Here we describe genetic assays commonly used to test interactions between transmembrane α-helices in their native membrane environment. These assays are based on the reconstitution of dimeric regulators allowing the control of expression of reporter genes. We provide detailed protocols for the TOXCAT and GALLEX assays used to monitor homotypic and heterotypic transmembrane helix-helix interactions.

Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.

Magnetically Oriented Bicelles with Monoalkylphosphocholines: Versatile Membrane Mimetics for Nuclear Magnetic Resonance Applications.

Bicelles (bilayered micelles) are model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- and short-chain phospholipids, usually dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because their composition and planar surface are similar to the native membrane environment. In this work, to improve the solubilization of membrane proteins and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family, of which dodecylphosphocholine (12PC) is known for its ability to solubilize membrane proteins. More specifically 12PC, tetradecyl- (14PC), and hexadecyl-PC (16PC) have been employed. To verify the possibility of making bicelles with different hydrophobic thicknesses to better accommodate membrane proteins, D14PC was also replaced by phospholipids with different alkyl chain lengths: dilauroyl-PC (D12PC), dipalmitoyl-PC (D16PC), distearoyl-PC (D18PC), and diarachidoyl-PC (D20PC). Results obtained by 31P solid-state nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) at several lipid-to-detergent molar ratios (q) and temperatures indicate that these new MAPCHO bicelles can be formed under a variety of conditions. The quality of their alignment is similar to that of classical bicelles, and the low critical micelle concentration (CMC) of the surfactants and their miscibility with phospholipids are likely to be advantageous for the reconstitution of membrane proteins.