Cotranslational assembly of membrane protein/nanoparticles in cell-free systems

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins.We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.     

Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.     

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Multidrug transporter MexB of Pseudomonas aeruginosa: overexpression, purification, and initial structural characterization

Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization.     

Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies

Cell-free expression techniques have emerged as promising tools for the production of membrane proteins for structural and functional analysis. Elimination of toxic effects and a variety of options to stabilize the synthesized proteins enable the synthesis of otherwise difficult to obtain proteins. Modifications in the reaction design result in preparative scale production rates of cell-free reactions and yield in milligram amounts of membrane proteins per one millilitre of reaction volume. A diverse selection of detergents can be supplied into the reaction system without inhibitory effects to the translation machinery. This offers the unique opportunity to produce a membrane protein directly into micelles of a detergent of choice. We present detailed protocols for the cell-free production of membrane proteins in different modes and we summarize the current knowledge of this technique. A special emphasize will be on the production of soluble and functionally folded membrane proteins in presence of suitable detergents. In addition, we will highlight the advantages of cell-free expression for the structural analysis of membrane proteins especially by liquid state nuclear magnetic resonance spectroscopy and we will discuss new strategies for structural approaches.     

Membrane protein assembly into Nanodiscs

Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins.     

Phase separation in the isolation and purification of membrane proteins

Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.     

Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange

Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.     

Simple screening method for improving membrane protein thermostability

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.     

Quality Control in Eukaryotic Membrane Protein Overproduction

The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies.     

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.     

Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.     

Crystal Structure of the Multidrug Exporter MexB from Pseudomonas aeruginosa

We report here the crystal structure of the Pseudomonas aeruginosa multidrug exporter MexB, an intensively studied member of the resistance-nodulation-cell division family of secondary active transporters, at 3.0 Å. MexB forms an asymmetric homotrimer where each subunit adopts a different conformation representing three snapshots of the transport cycle similar to the recently determined structures of its close homologue AcrB from Escherichia coli, so far the sole structurally characterized member of the superfamily. As for AcrB, the conformations of two subunits can be clearly assigned to either the binding step or the extrusion step in the transport process. Unexpectedly, a remarkable conformational shift in the third subunit is observed in MexB, which has potential implications for the assembly of the tripartite MexAB-OprM drug efflux system. Furthermore, an n-dodecyl-d-maltoside molecule was found bound to the internal multidrug-binding cavity, which might indicate that MexB binds and transports detergent molecules as substrates. As the only missing piece of the puzzle in the MexAB-OprM system, the X-ray structure of MexB completes the molecular picture of the major pump mediating intrinsic and acquired multidrug resistance in P. aeruginosa.     

Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-β-d-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.     

Reconstitution of purified membrane protein dimers in lipid nanodiscs with defined stoichiometry and orientation using a split GFP tether

Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors, and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here, we describe a technique to obtain purified homogeneous membrane protein dimers in nanodiscs using a split GFP (sGFP) tether. Complementary sGFP tags associate to tether the coexpressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative Western blotting and negative-stain EM. The sGFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody–conjugated resin. We used this method to purify a Frizzled-4 homodimer and a Frizzled-4/low-density lipoprotein receptor–related protein 6 heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.     

Heterologous expression and strategies for encapsulation of membrane-localized plant P450s

Heterologous expression of plant P450 proteins is critical for functional definitions of their enzymatic activities as well as for producing natural products whose biosyntheses involve P450s. Over the past decade and a half, several expression systems, using bacterial, yeast and insect cells, have been utilized successfully for expression of P450s from different plant species. Extensive optimizations in each system have focused on the improvement of expression levels, and the enhancement of the redox environment for catalytic activity. In this review, we discuss the strengths and limitations of each system, as well as recent developments and applications of each system. We also discuss the principles behind Nanodisc technology, which utilizes an amphipathic “membrane scaffold protein” (MSP) to stabilize the soluble membrane protein-containing nanometer diameter phospholipid bilayers, and its potential applications in plant P450 research.     

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.