Analysis Of Membrane Protein Complexes Research Articles

Ionic Liquid-Based Extraction System for In-Depth Analysis of Membrane Protein Complexes.

Limited by the rare efficient extraction system in extracting hydrophobic membrane protein complexes (MPCs) without compromising the stability of protein-protein interactions (PPIs), the in-depth functional study of MPCs has lagged far behind. In this study, the first systematic screening of ionic liquids (ILs) was performed and showed that triethylammonium acetate (TEAA) IL exhibited excellent performance in stabilizing PPIs, which was further confirmed by molecular docking simulations. By combining TEAA with the conventional detergent Nonidet P-40 (NP-40), a novel IL-based extraction system, i-TAN (TEAA IL with 1% NP-40), was proposed, which demonstrated superior performance in extracting and stabilizing MPCs, attributed to its larger size, more uniform distribution, and closer-to-neutral microenvironment of micelles. Extraction of MPCs with i-TAN allowed the confident identification of more hydrophobic EGFR-interacting proteins that are easily dissociated during the extraction process. Quantitative analysis of the difference in EGFR complexes between trastuzumab-sensitive and trastuzumab-resistant breast cancer cells provided comprehensive insights to understand the drug resistance mechanism, suggesting that i-TAN has great potential in interactomics and functional analysis of MPCs. This study provides a novel strategy for MPC extraction and downstream processing.

Controlled Bacterial Lysis for Electron Tomography of Native Cell Membranes

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ΦX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.

High resolution clear native electrophoresis is a good alternative to blue native electrophoresis for the characterization of the Escherichia coli membrane complexes

Blue native electrophoresis (BNE) has become the most popular method for the global analysis of membrane protein complexes. Although it has been shown to be very useful for that purpose, it can produce the dissociation of complexes with weak interactions and, due to the use of Coomassie Brilliant Blue, does not allow the subsequent application of fluorimetric and/or enzymatic techniques. Recently, we have successfully used the high resolution clear native electrophoresis (hrCNE) for the analysis of Neisseria meningitidis outer membrane porin complexes. The aim of this study was to determine the composition of the complexome of the Escherichia coli envelope by using hrCNE and to compare our results with those previously obtained using BNE. The bidimensional electrophoresis approaches used, hrCN/hrCNE and hrCN/SDS-PAGE, coupled to mass spectrometry allowed a detailed analysis of the complexome of E. coli membranes. For the first time, the three subunits of the formate dehydrogenase FDH-O were identified forming a single complex and hrCNE also allowed the identification of both the HflK and HflC proteins as components of the HflA complex. This technique also allowed us to suggest a relationship between OmpF and DLDH and, although OmpA is considered to be monomeric in vivo, we found this protein structured as homodimers. Thus hrCNE provides a good tool for future analyses of bacterial membrane proteins and complexes and is an important alternative to the commonly used BNE.

Formation and dissociation processes of gas-phase detergent micelles.

Growing interest in micelles to protect membrane complexes during the transition from solution to gas phase prompts a better understanding of their properties. We have used ion mobility mass spectrometry to separate and assign detergent clusters formed from the n-trimethylammonium bromide series of detergents. We show that cluster size is independent of detergent concentration in solution, increases with charge state, but surprisingly decreases with alkyl chain length. This relationship contradicts the thermodynamics of micelle formation in solution. However, the liquid drop model, which considers both the surface energy and charge, correlates extremely well with the experimental cluster size. To explore further the properties of gas-phase micelles, we have performed collision-induced dissociation on them during tandem mass spectrometry. We observed both sequential asymmetric charge separation and neutral evaporation from the precursor ion cluster. Interestingly, however, we also found markedly different dissociation pathways for the longer alkyl chain detergents, with significantly fewer intermediate ions formed than for those with a shorter alkyl chain. These experiments provide an essential foundation for understanding the process of the gas-phase analysis of membrane protein complexes. Moreover they imply valuable mechanistic details of the protection afforded to protein complexes by detergent clusters during gas-phase activation processes.

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.

Systematic Analysis of Native Membrane Protein Complexes inEscherichia coli

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

BackgroundClostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.ResultsA two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.ConclusionTwo dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.

Proteomics Analysis Reveals Overlapping Functions of Clustered Protocadherins

The three tandem-arrayed protocadherin (Pcdh) gene clusters, namely Pcdh-alpha, Pcdh-beta, and Pcdh-gamma, play important roles in the development of the vertebrate central nervous system. To gain insight into the molecular action of PCDHs, we performed a systematic proteomics analysis of PCDH-gamma-associated protein complexes. We identified a list of 154 non-redundant proteins in the PCDH-gamma complexes. This list includes nearly 30 members of clustered Pcdh-alpha, -beta, and -gamma families as core components of the complexes and additionally over 120 putative PCDH-associated proteins. We validated a selected subset of PCDH-gamma-associated proteins using specific antibodies. Analysis of the identities of PCDH-associated proteins showed that the majority of them overlap with the proteomic profile of postsynaptic density preparations. Further analysis of membrane protein complexes revealed that several validated PCDH-gamma-associated proteins exhibit reduced levels in Pcdh-gamma-deficient brain tissues. Therefore, PCDH-gamma s are required for the integrity of the complexes. However, the size of the overall complexes and the abundance of many other proteins remained unchanged, raising a possibility that PCDH-alphas and PCDH-betas might compensate for PCDH-gamma function in complex formation. As a test of this idea, RNA interference knockdown of both PCDH-alphas and PCDH-gamma s showed that PCDHs have redundant functions in regulating neuronal survival in the chicken spinal cord. Taken together, our data provide evidence that clustered PCDHs coexist in large protein complexes and have overlapping functions during vertebrate neural development.

Structural analysis of membrane protein complexes by single particle electron microscopy

Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7–10 Å resolution and 15–30 Å resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.

Analysis of Membrane Protein Complexes by Blue Native PAGE

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.

A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome.

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii.

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immuno-blotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-deficient (Deltaycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.