RESUMEN
Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli Additionally, transport was greatly reduced in the presence of ß-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport ß-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Mycobacterium smegmatis/metabolismo , Transportadores de Anión Orgánico/metabolismo , Sodio/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Regulación Bacteriana de la Expresión Génica , Metabolómica , Simulación del Acoplamiento Molecular , Transportadores de Anión Orgánico/genética , Filogenia , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/genéticaRESUMEN
Glutamate transporters play important roles in bacteria, archaea and eukaryotes. Their function in the mammalian central nervous system is essential for preventing excitotoxicity, and their dysregulation is implicated in many diseases, such as epilepsy and Alzheimer's. Elucidating their transport mechanism would further the understanding of these transporters and promote drug design as they provide compelling targets for understanding the pathophysiology of diseases and may have a direct role in the treatment of conditions involving glutamate excitotoxicity. This review outlines the insights into the transport cycle, uncoupled chloride conductance and modulation, as well as identifying areas that require further investigation.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Archaea/metabolismo , Sistema de Transporte de Aminoácidos X-AG/química , Ácido Aspártico/metabolismo , Sitios de Unión , Cloruros/metabolismo , Ácido Glutámico/metabolismo , Humanos , Potasio/metabolismo , Conformación Proteica , Sodio/metabolismoRESUMEN
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.
Asunto(s)
Microscopía por Crioelectrón/métodos , Membrana Dobles de Lípidos/química , Maleatos/química , Proteínas de la Membrana/química , Poliestirenos/química , Cristalografía por Rayos X , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/ultraestructura , Conformación Proteica , Multimerización de Proteína , Proteolípidos/química , Proteolípidos/ultraestructuraRESUMEN
Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.
Asunto(s)
Proteínas de Escherichia coli/química , Membrana Dobles de Lípidos/química , Maleatos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Poliestirenos/química , Proteínas Recombinantes/química , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Microscopía Electrónica/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Coloración y Etiquetado/métodosRESUMEN
Glutamate transporters are responsible for uptake of the neurotransmitter glutamate in mammalian central nervous systems. Their archaeal homologue GltPh, an aspartate transporter isolated from Pyrococcus horikoshii, has been the focus of extensive studies through crystallography, MD simulations and single-molecule FRET (smFRET). Here, we summarize the recent research progress on GltPh, in the hope of gaining some insights into the transport mechanism of this aspartate transporter.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Pyrococcus horikoshii/metabolismo , Ácido Aspártico/metabolismo , Transporte Biológico , Conformación Proteica , Especificidad por SustratoRESUMEN
Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Membranas Artificiales , Métodos , Bacterias/metabolismo , Eucariontes/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiologíaRESUMEN
Peroxisomes are organelles that perform diverse metabolic functions in different organisms, but a common function is ß-oxidation of a variety of long chain aliphatic, branched, and aromatic carboxylic acids. Import of substrates into peroxisomes for ß-oxidation is mediated by ATP binding cassette (ABC) transporter proteins of subfamily D, which includes the human adrenoleukodystropy protein (ALDP) defective in X-linked adrenoleukodystrophy (X-ALD). Whether substrates are transported as CoA esters or free acids has been a matter of debate. Using COMATOSE (CTS), a plant representative of the ABCD family, we demonstrate that there is a functional and physical interaction between the ABC transporter and the peroxisomal long chain acyl-CoA synthetases (LACS)6 and -7. We expressed recombinant CTS in insect cells and showed that membranes from infected cells possess fatty acyl-CoA thioesterase activity, which is stimulated by ATP. A mutant, in which Serine 810 is replaced by asparagine (S810N) is defective in fatty acid degradation in vivo, retains ATPase activity but has strongly reduced thioesterase activity, providing strong evidence for the biological relevance of this activity. Thus, CTS, and most likely the other ABCD family members, represent rare examples of polytopic membrane proteins with an intrinsic additional enzymatic function that may regulate the entry of substrates into the ß-oxidation pathway. The cleavage of CoA raises questions about the side of the membrane where this occurs and this is discussed in the context of the peroxisomal coenzyme A (CoA) budget.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Tioléster Hidrolasas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Acilcoenzima A/metabolismo , Adenosina Trifosfatasas , Sustitución de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico Activo , Coenzima A Ligasas/metabolismo , Proteínas de Transporte de Ácidos Grasos/genética , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Peroxisomas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tioléster Hidrolasas/genéticaRESUMEN
In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated â¼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfatasas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Hemo/química , Multimerización de Proteína , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Transporte Biológico Activo/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hemo/genética , Hemo/metabolismo , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.
Asunto(s)
Modelos Moleculares , Shewanella/genética , Simportadores/ultraestructura , Sitios de Unión/genética , Cristalografía , Transportador de Péptidos 1 , Simportadores/genéticaRESUMEN
Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (â¼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Nucleósidos/química , Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/química , Nucleósidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Mutación , Proteínas de Transporte de Nucleósidos/genética , Nucleósidos/genética , Alineación de Secuencia , Simportadores/química , Simportadores/genética , Simportadores/metabolismoRESUMEN
Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/química , Canales Iónicos/química , Proteínas de Transporte de Membrana/química , Urea/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Canales Iónicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Urea/químicaRESUMEN
Contamination with the multidrug transporter AcrB represents a potential pitfall in the structural analysis of recombinant membrane proteins expressed in Escherichia coli, especially when high-throughput approaches are adopted. This can be a particular problem in two-dimensional (2-D) crystallization for electron cryomicroscopy since individual crystals are too small for compositional analysis. Using a broad 'sparse matrix' of buffer conditions typically used in 2-D crystallization, we have identified at least eight unique crystal forms of AcrB. Reference to images and projection maps of these different forms can greatly facilitate the early identification of false leads in 2-D crystallization trials of other membrane proteins of interest. We illustrate the usefulness of such data by highlighting two studies of membrane proteins in our laboratories. We show in one case (a bacterial sodium channel, NaChBac) how early crystallization 'hits' could be attributed to contaminating AcrB by comparison against our AcrB crystal image database. In a second case, involving a member of the monovalent cation/proton antiporter-1 family (MPSIL0171), a comparison with the observed AcrB crystal forms allowed easy identification of reconstituted AcrB particles, greatly facilitating the eventual purification and crystallization of the correct protein in pure form as ordered helical arrays. Our database of AcrB crystal images will be of general use in assisting future 2-D crystallization studies of other membrane proteins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Canales de Sodio/química , Cationes Monovalentes/química , Cristalización/métodos , Cristalografía por Rayos XAsunto(s)
Bioquímica/historia , Inglaterra , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM.
Asunto(s)
Microscopía por Crioelectrón/métodos , Cicloparafinas/química , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/aislamiento & purificación , Polímeros/química , Microscopía por Crioelectrón/instrumentaciónRESUMEN
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.
RESUMEN
A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
Asunto(s)
Cromatografía de Afinidad/métodos , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Animales , Escherichia coli/crecimiento & desarrollo , Vectores Genéticos , Humanos , Células Sf9RESUMEN
Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.
RESUMEN
Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.
Asunto(s)
Maleatos/química , Lípidos de la Membrana/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Poliestirenos/química , Microscopía por Crioelectrón/métodos , Detergentes , Escherichia coli/química , Proteínas de Escherichia coli/química , Membrana Dobles de Lípidos/química , Gotas Lipídicas/química , Espectrometría de Masas/métodos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/química , Polímeros/químicaRESUMEN
The preparation of cell membranes by ultracentrifugation of bacterial cell lysates, a pre-requisite for the purification of over-expressed membrane proteins, is both time-consuming and difficult to perform on a large scale. To overcome this bottleneck in the structural investigation of such proteins in the UK Membrane Protein Structure Initiative, we have investigated the alternative use of tangential flow filtration for preparation of membranes from Escherichia coli. This method proved to be superior to the conventional use of ultracentrifuges both in speed and in yield of membrane protein. Moreover, it could more readily be scaled up to process larger quantities of bacterial cells. Comparison of the purity and monodispersity of an over-expressed membrane protein purified from conventionally-prepared membranes and from membranes prepared by filtration revealed no substantial differences. The approach described should therefore be of general use for membrane protein preparation for a wide range of applications, including both structural and functional studies.