Trimeric Membrane Protein Research Articles

PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters

Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K+ ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH3) or NH3 coupled with an H+ are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K+ AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH4+ deprotonation during passage, substantially transported K+ in addition to NH4+ Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH4+ deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K+ We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease.

The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.

Multimeric Soluble 4-1BBL as a T Cell Stimulator for Adoptive Immunotherapy.

Abstract Members of the TNF SuperFamily of ligands (TNFSFs) have significant potential as immuno-oncology agents. The TNFSFs are trimeric membrane proteins that can be cleaved into soluble single trimers. While the soluble single trimers can be easily prepared and studied, they have little or no activity in vivo. This deficiency is caused by the need to cluster their cognate receptors in the plane of the membrane in order to induce a supramolecular signaling complex on the cytoplasmic side of the plasma membrane. For the TNFSF ligands, this requires that they be used as many-trimer multimers that mimic the natural expression of many trimers on the surface of stimulating cells. To meet this need, we prepared fusion proteins comprised of the extracellular domains of TNFSF ligands joined to a natural protein that provides a multimerization scaffold. When surfactant protein D (SP-D) is used as a scaffold, the result is a 4-trimer TNFSF ligand product (UltraLigand™). Our published studies have described such multimeric forms of CD40L, OX40L, GITRL, CD27L/CD70, BAFF, RANKL, and TRAIL and shown that they are highly active in vitro and in vivo. As an extension of this work, 4-trimer forms of murine and human 4-1BBL (CD137L, TNFSF9) were constructed and expressed in CHO cells. As a co-stimulatory molecule, SP-D-4-1BBL (Ultra4-1BBL) activated both CD4+ and CD8+ T cells and promoted the growth of NK cells in vitro. Given the interest in 4-1BB (CD137) as a marker of therapeutically effective tumor-infiltrating lymphocytes (TILs), SP-D-4-1BBL should be a useful should be a useful growth factor for TIL manufacturing and T cell culturing in general.

Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity

Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755–1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.

Systematic solution to homo-oligomeric structures determined by NMR.

Protein structure determination by NMR has predominantly relied on simulated annealing-based conformational search for a converged fold using primarily distance constraints, including constraints derived from nuclear Overhauser effects, paramagnetic relaxation enhancement, and cysteine crosslinkings. Although there is no guarantee that the converged fold represents the global minimum of the conformational space, it is generally accepted that good convergence is synonymous to the global minimum. Here, we show such a criterion breaks down in the presence of large numbers of ambiguous constraints from NMR experiments on homo-oligomeric protein complexes. A systematic evaluation of the conformational solutions that satisfy the NMR constraints of a trimeric membrane protein, DAGK, reveals 9 distinct folds, including the reported NMR and crystal structures. This result highlights the fundamental limitation of global fold determination for homo-oligomeric proteins using ambiguous distance constraints and provides a systematic solution for exhaustive enumeration of all satisfying solutions.

Structural Insights for the Optimization of Dihydropyrimidin-2(1H)-one Based mPGES-1 Inhibitors.

The recently crystallized structure of microsomal prostaglandin E2 synthase 1 (mPGES-1) in complex with the inhibitor LVJ (PDB code: 4BPM) offered new structural information for the optimization of the previously identified lead compound 1 (IC50 = 4.16 ± 0.47 μM), which contains the privileged dihydropyrimidin-2-one chemical core. Systematic optimization of 1, through accurate structure-based design, provided compound 4 with a 10-fold improved mPGES-1 inhibitory activity (IC50 = 0.41 ± 0.02 μM). Here we highlight the optimal scaffold decoration pattern of 4 and propose a three-dimensional model for the interaction with this complex trimeric membrane protein. The reported computational insights, together with the accessible one-pot synthetic procedure, stimulate for the generation of further potent dihydropyrimidine-based mPGES-1 inhibitors.

Molecular Dynamics Simulation Studies of Interactions of E. coli-K12 with OmpF in Outer Membranes: Effects of LPS Structures on Monoclonal Antibodies Binding

Gram-negative bacterial outer membrane, which is a highly asymmetric lipid bilayer, is composed of an outer membrane protein along with phospholipids (PLs) forming the inner leaflet and lipopolysaccharides (LPSs) forming the outer leaflet. Outer membrane protein F (OmpF) porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules such as nutrients and waste products across the outer membrane of Escherichia coli (E. coli). Here, we report the structural properties of a model of the E. coli-K12 outer membrane and its interaction with OmpF using molecular dynamics simulations. Immunochemical and other experiments suggested several cell-surface exposed epitopes (antibody binding sites), which were recognized by various monoclonal antibodies (MAbs). Molecular details of interaction between LPS core sugars and surface of OmpF suggest the importance of LPS core sugars in shielding of these epitopes. Results are compared with experimental evidence, which showed that with shortening of the LPS core sugars (rfa E. coli-K-12 mutants), the number of MAbs, that recognized porin surface epitopes, increased sequentially. To check the stabilizing effect of LPS structure on the extracellular loop conformations, results of interaction between OmpF and asymmetric E. coli-K12 bilayer are compared to those from OmpF simulations in a DMPC bilayer. In addition, overall membrane properties such as, electron density profiles, per-lipid surface area of each lipid type, and chain order parameters are compared with E.coli-K12 outer membrane in absence of OmpF. These results are also compared to our previous molecular dynamics results with E. coli-R1 outer membrane with OmpLA. Overall, membrane structural properties are comparable, however, individual lipid properties showed slight variations.

Multimeric forms of 4-1BBL as stimulators of T cells for adoptive immunotherapy

Meeting abstracts Members of the TNF SuperFamily of ligands (TNFSFs) have significant potential as immuno-oncology therapeutic agents. The TNFSFs are trimeric membrane proteins that can be cleaved into soluble single trimers. While the soluble single trimers can be easily prepared and studied, they

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB.

The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments

Förster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ∼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.

ChemInform Abstract: Recent Advances in Solid‐State Nuclear Magnetic Resonance Techniques to Quantify Biomolecular Dynamics

AbstractReview: 52 refs.

The FRET Signatures of Non-Interacting Proteins in Cellular Membranes

Forster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of 100 A. This effect does not reflect specific interactions in the membrane and is rarely appreciated in the field, despite the fact that its magnitude can be significant. Here we develop a comprehensive computational description of proximity FRET, simulating the cases of proximity FRET when the fluorescent proteins are used to tag monomeric, dimeric, trimeric and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibrium. We also perform rigorous experimental measurements of this effect by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between YFP and m-Cherry-tagged versions of these receptors in plasma membrane derived vesicles, as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here should bring much-needed rigor in FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics.

The emergence of solid-state NMR (SSNMR) techniques over the last decade has greatly expanded the range of accessible biomolecules amenable to structural study.1-2 These methods were developed and first applied to a variety of microcrystalline model proteins including BPTI,3 SH3,4-5 ubiquitin,6-7 kaliotoxin,8 and GB1.9-11 These studies demonstrated that individual 13C, 15N and 1H sites throughout the entire protein could be uniquely resolved and assigned for purposes of high-resolution structure determination. Whereas traditional solid-state NMR approaches required site-specific isotopic labeling, the new developments enable entire proteins to be examined by resolving signals in multiple dimensions. These capabilities also permit the measurement of dynamic parameters through correlation spectroscopy, which increases the throughput, sensitivity, reliability and reproducibility of such measurements. A major advantage of SSNMR is that a large range of sample conditions and types can be examined, including many that are not accessible to solution NMR methods, such as membrane proteins and fibrils in addition to microcrystals. Recent examples include fibrils of the HET-s prion,12 high molecular weight oligomers of αB crystallin,13 the tetrameric assembly of the M2 peptide with bound amantadine drug in physiologically relevant bilayers,14 the trimeric membrane protein YadA,15 the photoreceptor proteorhodopsin,16 fibrils of the core domain of transthyretin,17 and beta-amyloid plaques derived from the brains of Alzheimer's disease patients.18 Such studies have opened up new avenues for exploration of fundamental biophysics and biochemistry, and yielded insights into clinically relevant events in membranes and aggregated states not accessible to solution NMR or X-ray crystallography. Moreover, SSNMR experiments are applicable over a larger range of temperatures than can be accessed by solution NMR19 and thereby enable a continuum of experimental conditions from physiological temperatures to the cryogenic temperatures at which most crystal structures are solved. Another advantage of NMR methods in general is that they are non-perturbing and do not require covalent modification of the protein. Thus, the dynamic information procured from such experiments has the potential to accurately represent the native protein behavior. This review focuses on developments over the last five years, regarding methods and applications of dynamics studies by solid-state NMR over timescales ranging from nanoseconds to days.

P2X Receptor Intermediate Activation States Have Altered Nucleotide Selectivity

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

Molecular insights into type I secretion systems

Type 1 secretion systems are one of the main machineries in Gram-negative bacteria involved in the secretion of a wide range of substrates from the cytoplasm across the inner and outer membrane in one step to the extracellular space. The range of substrates varies from small proteins up to large surface layer proteins of about 900 kDa. Most of the substrates have a non-cleavable C-terminal secretion signal and so-called GG repeats that are able to bind calcium ions. The translocator complex is composed of a trimeric outer membrane protein that provides a pore in the outer membrane. A multimeric membrane fusion protein spans the periplasm and forms a continuous channel connecting the outer membrane protein with a dimeric ATP-binding cassette transporter in the inner membrane. The ATP-binding cassette-transporter is thought to form a channel through the inner membrane and energizes the transport process. This review will provide a detailed view of the components of the translocator and will summarize structural as well as functional data.

Catalytic Characterization of Human Microsomal Glutathione S-Transferase 2: Identification of Rate-Limiting Steps

Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S exhibit distinct catalytic and mechanistic properties, reflecting adaptation to broad and specific physiological functions, respectively.

P2X7 Receptor Channels Allow Direct Permeation of Nanometer-Sized Dyes

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.

Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies.

The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.