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Sample records for membrane protein gene

  1. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Treesearch

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  2. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  3. Trypanosoma cruzi modulates gene expression of plasma membrane repair-related proteins.

    PubMed

    Brígido, Rebecca Tavares E Silva; Tavares, Paula Cristina Brígido; Santos, Marlus Alves Dos; Santos, Júlia de Gouveia; Souza, Maria Aparecida de; Goulart, Isabela Maria Bernardes; Silva, Claudio Vieira da

    2017-10-01

    Plasma membrane injury and repair is particularly prevalent in muscle cells. Here, we aimed to verify dysferlin, acid sphingomyelinase and transcriptional factor EB gene expression during Trypanosoma cruzi infection in vitro and in vivo. Our results showed that the parasite modulates gene expression of these proteins in a way dependent on the number of plasma membrane interacting parasites and in a rapamycin-sensitive manner. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Use of gene fusions to study outer membrane protein localization in Escherichia coli.

    PubMed Central

    Silhavy, T J; Shuman, H A; Beckwith, J; Schwartz, M

    1977-01-01

    Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport. Images PMID:414221

  5. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis.

    PubMed

    Town, M; Jean, G; Cherqui, S; Attard, M; Forestier, L; Whitmore, S A; Callen, D F; Gribouval, O; Broyer, M; Bates, G P; van't Hoff, W; Antignac, C

    1998-04-01

    Nephropathic cystinosis, an autosomal recessive disorder resulting from defective lysosomal transport of cystine, is the most common inherited cause of renal Fanconi syndrome. The cystinosis gene has been mapped to chromosome 17p13. We found that the locus D17S829 was homozygously deleted in 23 out of 70 patients, and identified a novel gene, CTNS, which mapped to the deletion interval. CTNS encodes an integral membrane protein, cystinosin, with features of a lysosomal membrane protein. Eleven different mutations, all predicted to cause loss of function of the protein, were found to segregate with the disorder.

  6. Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

    PubMed

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo

    2005-12-01

    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

  7. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  8. Evolutionary origins of membrane proteins

    NASA Astrophysics Data System (ADS)

    Mulkidjanian, Armen Y.; Galperin, Michael Y.

    Although the genes that encode membrane proteins make about 30% of the sequenced genomes, the evolution of membrane proteins and their origins are still poorly understood. Here we address this topic by taking a closer look at those membrane proteins the ancestors of which were present in the Last Universal Common Ancestor, and in particular, the F/V-type rotating ATPases. Reconstruction of their evolutionary history provides hints for understanding not only the origin of membrane proteins, but also of membranes themselves. We argue that the evolution of biological membranes could occur as a process of coevolution of lipid bilayers and membrane proteins, where the increase in the ion-tightness of the membrane bilayer may have been accompanied by a transition from amphiphilic, pore-forming membrane proteins to highly hydrophobic integral membrane complexes.

  9. The Rh protein family: gene evolution, membrane biology, and disease association.

    PubMed

    Huang, Cheng-Han; Ye, Mao

    2010-04-01

    The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor's involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.

  10. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  11. Mycorrhizal fungi influence on silver uptake and membrane protein gene expression following silver nanoparticle exposure

    NASA Astrophysics Data System (ADS)

    Noori, Azam; White, Jason C.; Newman, Lee A.

    2017-02-01

    The rapid growth of nanotechnology and the high demand for nanomaterial use have greatly increased the risk of particle release into the environment. Understanding nanomaterial interactions with crop species and their associated microorganisms is critical to food safety and security. In the current study, tomato was inoculated with mycorrhizal fungi and subsequently exposed to 12, 24, or 36 mg/kg of 2- or 15-nm silver nanoparticles (Ag-NPs). Mycorrhizal (M) and non-mycorrhizal (NM) tomatoes exposed to 36 mg/kg of 2-nm Ag-NPs accumulated 1300 and 1600 μg/g silver in their tissues, respectively. Mycorrhizal plants accumulated 14% less silver compared to non-mycorrhizal plants. To begin to understand the mechanisms by which plants accumulate NPs, the expression of two aquaporin channel genes, the plasma membrane intrinsic protein (PIP) and the tonoplast membrane intrinsic protein (TIP), and one potassium channel (KC) gene were studied. In non-mycorrhizal plants, the expression of KC, PIP, and TIP was eight, five, and nine times higher than the control, respectively. These expressions for mycorrhizal plants were 5.8, 3.5, and 2 times higher than controls, respectively. The expression of KC and PIP, which are located on the plasma membrane, was 3.5 and 2.5, respectively, times higher than TIP, which is located on the tonoplast. PIP expression was significantly higher in NM tomatoes exposed to 12 mg/kg of 2-nm Ag-NPs compared to M plants. These results show that mycorrhizal colonization decreases Ag accumulation in NP-exposed plants and also moderates changes in expression level of membrane transport proteins.

  12. [Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae].

    PubMed

    Wang, Yan; Zhang, Lei; Zhang, Li; Wang, Han

    2011-07-01

    To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB. A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting. Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli. The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

  13. Split-Doa10: a naturally split polytopic eukaryotic membrane protein generated by fission of a nuclear gene.

    PubMed

    Stuerner, Elisabeth; Kuraku, Shigehiro; Hochstrasser, Mark; Kreft, Stefan G

    2012-01-01

    Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12-16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.

  14. Identification of the Salmonella enterica damX Gene Product, an Inner Membrane Protein Involved in Bile Resistance▿ †

    PubMed Central

    López-Garrido, Javier; Cheng, Nancy; García-Quintanilla, Fátima; García-del Portillo, Francisco; Casadesús, Josep

    2010-01-01

    The damX gene product of Salmonella enterica serovar Typhimurium is a protein located in the inner membrane. DamX migrates as a 70-kDa protein in SDS-PAGE even though the predicted protein size is 46 kDa. Synthesis of DamX protein occurs in both exponential- and stationary-phase cultures. Disruption of damX causes severe sensitivity to bile. Lack of the outer membrane protein AsmA suppresses bile sensitivity in Salmonella damX mutants. PMID:19948803

  15. Structure and promoter analysis of the mouse membrane-bound transferrin-like protein (MTf) gene.

    PubMed

    Nakamasu, K; Kawamoto, T; Yoshida, E; Noshiro, M; Matsuda, Y; Kato, Y

    2001-03-01

    Recently, we purified membrane-bound transferrin-like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem. 256, 503--509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning approximately 26 kb and consisting of 16 exons. The transcription-initiation site was located 157 bp upstream from the translation-start codon, and a TATA box was not found in the 5' flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK-MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5' flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5' deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtained with primary chondrocytes suggest that the regions between -693 and -444 and between -1635 and -1213 contain positive and negative cis-acting elements, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in

  16. The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation

    PubMed Central

    1995-01-01

    We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle. PMID:7844145

  17. Sequence analysis of the major outer membrane protein gene of Chlamydia pneumoniae.

    PubMed Central

    Perez Melgosa, M; Kuo, C C; Campbell, L A

    1991-01-01

    Compared with the major outer membrane proteins (MOMPs) of the other chlamydial species, the Chlamydia pneumoniae MOMP appears to be less antigenically complex, and as determined by immunoblot analysis, it does not appear to be the immunodominant antigen recognized during infection. Nucleotide sequence analysis of the C. pneumoniae MOMP gene (ompA) revealed that it consisted of a 1,167-base open reading frame with an inferred 39,344-dalton mature protein of 366 amino acids plus a 23-amino-acid leader sequence. A ribosomal-binding site was located in the 5' upstream region, and two stop codons followed by an 11-base dyad forming a stable stem-loop structure were identified. This sequence shares 68 and 71% DNA sequence homology to the Chlamydia trachomatis serovar L2 and Chlamydia psittaci ovine abortion agent MOMP genes, respectively. Interspecies alignment identified regions, corresponding to the variable domains, which share little sequence similarity with the other chlamydial MOMPs. All seven cysteines conserved in the C. trachomatis and C. psittaci MOMPs, which are involved in the formation of disulfide cross-linkages, are found in the C. pneumoniae MOMP. PMID:1840574

  18. Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum.

    PubMed

    Takaoka, N; Fukuzawa, M; Saito, T; Sakaitani, T; Ochiai, H

    1999-10-28

    We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position -47 to -37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position -3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the beta-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.

  19. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene.

    PubMed Central

    Duchêne, M; Schweizer, A; Lottspeich, F; Krauss, G; Marget, M; Vogel, K; von Specht, B U; Domdey, H

    1988-01-01

    Porin F is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. It forms water-filled pores of variable size. Porin F is a candidate for a vaccine against P. aeruginosa because it antigenically cross-reacts in all serotype strains of the International Antigenic Typing Scheme. We have isolated the gene for porin F from a lambda EMBL3 bacteriophage library by using oligodeoxynucleotide hybridization probes and have determined its nucleotide sequence. Different peptide sequences obtained from isolated porin F confirmed the deduced protein sequence. The mature protein consists of 326 amino acid residues and has a molecular weight of 35,250. The precursor contains an N-terminal signal peptide of 24 amino acid residues. S1 protection and primer extension experiments, together with Northern (RNA) blots, indicate that the mRNA coding for porin F is monocistronic with short untranslated regions of about 58 bases at the 5' end and about 47 bases at the 3' end. The sequences in the -10 and -35 regions upstream of the transcriptional start site are closely related to the Escherichia coli promoter consensus sequences, which explains why the porin F gene is expressed in E. coli under the control of its own promoter. The amino acid sequence of porin F is not homologous to the different E. coli porins OmpF, OmpC, LamB, and PhoE. On the other hand, a highly homologous region of 30 amino acids between the OmpA proteins of different enteric bacteria and porin F of P. aeruginosa was detected. The core region of the homology to E. coli OmpA had 11 of 12 amino acid residues in common. Images PMID:2447060

  20. Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO.

    PubMed Central

    Bally, M; Ball, G; Badere, A; Lazdunski, A

    1991-01-01

    xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria. Images PMID:1898929

  1. Bacteriophage T5 gene A2 protein alters the outer membrane of Escherichia coli.

    PubMed Central

    Snyder, C E

    1984-01-01

    Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 protein was obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis, and in vitro binding studies with purified gene A2 protein. The results suggested that the T5 gene A2 protein perturbs the host envelope as it functions to promote DNA transfer. Images PMID:6389511

  2. Nuclear membrane protein emerin: roles in gene regulation, actin dynamics and human disease.

    PubMed

    Wilson, Katherine L; Holaska, James M; Montes de Oca, Rocio; Tifft, Kathryn; Zastrow, Michael; Segura-Totten, Miriam; Mansharamani, Malini; Bengtsson, Luiza

    2005-01-01

    Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.

  3. impA, a Gene Coding for an Inner Membrane Protein, Influences Colonial Morphology of Actinobacillus actinomycetemcomitans

    PubMed Central

    Mintz, Keith P.; Fives-Taylor, Paula M.

    2000-01-01

    Directed mutagenesis of a gene coding for a membrane protein of the periodontopathogen Actinobacillus actinomycetemcomitans was achieved by conjugation. The gene was disrupted by insertion of an antibiotic cassette into a unique endonuclease restriction sequence engineered by inverse PCR. The disrupted gene was cloned into a conjugative plasmid and transferred from Escherichia coli to A. actinomycetemcomitans. The allelic replacement mutation resulted in the loss of a 22-kDa inner membrane protein. The loss of this protein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in impA was a change in the pattern of growth of the mutant bacteria in broth cultures. The progenitor bacteria grew as a homogeneous suspension of cells compared to a granular, autoaggregating adherent cell population described for the mutant bacteria. These data suggest that ImpA may play a regulatory role or be directly involved in protein(s) that are exported and associated with colony variations in A. actinomycetemcomitans. PMID:11083768

  4. Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins.

    PubMed

    Lange, Andreas; Kistler, Claudia; Jutzi, Tanja B; Bazhin, Alexandr V; Klemke, Claus Detlev; Schadendorf, Dirk; Eichmüller, Stefan B

    2009-06-01

    The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.

  5. Two genes encode the major membrane-associated protein of methanol-induced peroxisomes from Candida boidinii.

    PubMed

    Garrard, L J; Goodman, J M

    1989-08-15

    A massive proliferation of peroxisomes occurs in the yeast Candida boidinii when methanol is utilized as the sole carbon source; these peroxisomes contain the enzymes which catalyze the initial steps of methanol utilization. The most abundant peroxisomal membrane-associated protein has an apparent molecular mass of 20 kDa and is termed PMP20. We report the isolation of two genes that encode very similar forms of PMP20; this is the first report of genes that encode proteins associated with peroxisomal membranes. Southern analysis demonstrates that the two genes are on different loci, although there are several homologous regions of both 5'- and 3'-untranslated sequence. One of the areas of 5' homology is within the untranslated region of the mRNA. Within the coding region there are 35 base differences between the two genes that are reflected in only five amino acid differences. The mRNAs representing both genes of PMP20 are induced in cells grown in methanol-containing medium and are below detection in cells grown in glucose. S1 nuclease protection analysis indicates that there is a 2.5-fold difference in mRNA expression between the two genes when induced. The predicted sequences of both PMP20 genes show the absence of a cleaved amino-terminal leader sequence and the presence of only 1 cysteine residue. In agreement with previous biochemical data suggesting a peripheral association of this protein with the membrane (Goodman, J. M., Maher, J., Silver, P. A., Pacifico, A., and Sanders, D. (1986) J. Biol. Chem. 261, 3464-3468), there are no obvious membrane spanning regions predicted in the sequences. Both PMP20 gene products contain the carboxyl-terminal sequence AKL, similar to the putative SKL peroxisomal sorting sequence (Gould, S. J., Keller, G.-A., and Subramani, S. (1988) J. Cell Biol. 107, 897-905).

  6. Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X

    PubMed Central

    MENG, Xianrong; LIU, Xueling; ZHANG, Liyuan; HOU, Bo; LI, Binyou; TAN, Chen; LI, Zili; ZHOU, Rui; LI, Shaowen

    2016-01-01

    Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant. PMID:27149893

  7. Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

    PubMed

    Chaudhuri, Pallab; Kumar, S Vinoth; Prasad, Rajeev; Srivastava, S K; Yadav, M P

    2005-09-01

    Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

  8. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    DOEpatents

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  9. Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

    PubMed Central

    Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

    1992-01-01

    Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

  10. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  11. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  12. Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli ▿

    PubMed Central

    Skretas, Georgios; Georgiou, George

    2010-01-01

    The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme β-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production. PMID:20639362

  13. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus.

    PubMed Central

    Ficht, T A; Bearden, S W; Sowa, B A; Adams, L G

    1989-01-01

    The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When

  14. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    PubMed

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  15. [Cloning of major outer membrane protein gene of Legionella pneumophila and detection of its expression in prokaryotic cell].

    PubMed

    Zhang, Lei; Chen, Jianping; Wang, Tao; Zhang, Li; Tian, Yu

    2006-04-01

    In this study, the ompS gene, a major outer membrane protein gene of Legionella pneumophila, was obtained from the DNA of Legionella pneumophila by PCR. The gene was cloned into prokaryotic expressional plasmid pUC18 to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli strain BL21. The identification was made by means of restriction enzyme analysis, polymerase chain reaction, DNA sequencing analysis, SDS--polyacrylamine gel electrophoresis analysis and Western blot. The results showed that the ompS gene of 914 bp was amplified from Legionella pneumophila DNA, the recombinant plasmid pLPompS was constructed and its expression in prokaryotic cell was detected successfully.

  16. Comparative analysis of the structures of the outer membrane protein P1 genes from major clones of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Grass, S; Einhorn, M; Bailey, C; Newell, C

    1989-01-01

    P1 outer membrane proteins from Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants. Images PMID:2572549

  17. Laboratory information management system for membrane protein structure initiative--from gene to crystal.

    PubMed

    Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

    2008-12-01

    Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

  18. The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein.

    PubMed Central

    Robbins, K C; Leal, F; Pierce, J H; Aaronson, S A

    1985-01-01

    The v-sis transforming gene encodes the woolly monkey homologue of human platelet-derived growth factor (PDGF) polypeptide 2. After its synthesis on membrane bound polyribosomes, the glycosylated precursor dimerizes in the endoplasmic reticulum and travels through the Golgi apparatus. At the cell periphery, the precursor is processed to yield a dimer structurally analogous to biologically active PDGF. Small amounts of two incompletely processed forms are detectable in tissue culture fluids of simian sarcoma virus (SSV) transformants. However, the vast majority remains cell associated. Thus, this growth factor-related transforming gene product is not a classical secretory protein. These findings define possible cellular locations where the transforming activity of the sis-PDGF-2 protein may be exerted. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 11. PMID:2992941

  19. A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.

    PubMed Central

    Maniak, M; Nellen, W

    1988-01-01

    We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein. Images PMID:3336356

  20. Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

    PubMed Central

    Dean, G E; Macnab, R M; Stader, J; Matsumura, P; Burks, C

    1984-01-01

    The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix. PMID:6090403

  1. The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system.

    PubMed

    Jin, Min; Goldenring, James R

    2006-06-01

    Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

  2. The GmFWL1 (FW2-2-like) nodulation gene encodes a plasma membrane microdomain-associated protein.

    PubMed

    Qiao, Zhenzhen; Brechenmacher, Laurent; Smith, Benjamin; Strout, Gregory W; Mangin, William; Taylor, Christopher; Russell, Scott D; Stacey, Gary; Libault, Marc

    2017-08-01

    The soybean gene GmFWL1 (FW2-2-like1) belongs to a plant-specific family that includes the tomato FW2-2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2-2-like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain-associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process. © 2017 John Wiley & Sons Ltd.

  3. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  4. Cloning and characterization of CSP37, a novel gene encoding a putative membrane protein of Candida albicans.

    PubMed Central

    Sentandreu, M; Nieto, A; Iborra, A; Elorza, M V; Ponton, J; Fonzi, W A; Sentandreu, R

    1997-01-01

    In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. The gene was isolated by immunological screening of a DNA library constructed from mycelial cells with a polyclonal serum raised against cell walls of this morphology. Analysis of the nucleotide sequence of a corresponding genomic DNA fragment revealed a single open reading frame which encodes a predicted protein of 321 amino acids with no significant homology to others in the databases. Disruption of the CSP37 gene by the method described by Fonzi and Irwin (Genetics 134:717-728, 1993) eliminated expression of the Csp37 protein. The mutant strains showed no apparent defect in cell viability, growth, or cell wall assembly but displayed attenuated virulence in systemic infections induced in mice and reduced the ability to adhere to polystyrene. PMID:9244249

  5. The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

    PubMed Central

    Waterham, H R; de Vries, Y; Russel, K A; Xie, W; Veenhuis, M; Cregg, J M

    1996-01-01

    We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1. PMID:8628321

  6. C-erbB-2 gene product, a membrane protein commonly expressed on human fetal epithelial cells.

    PubMed

    Mori, S; Akiyama, T; Yamada, Y; Morishita, Y; Sugawara, I; Toyoshima, K; Yamamoto, T

    1989-07-01

    C-erbB-2 is a human proto-oncogene which has homologies with the well known proto-oncogene c-erbB. The c-erbB-2 gene is amplified and overexpressed in some human adenocarcinomas. Its expression, in terms of RNA levels in normal human fetal kidney, lung and liver, has also been reported. In the present study, various fetal tissues from three human abortuses obtained at 9, 14 and 24 weeks of gestation, were studied immunohistologically by the ABC method and immunochemically by Western blot analysis for the distribution of c-erbB-2 gene product at the protein level. A polyclonal antibody raised in rabbit by immunization with a synthetic polypeptide corresponding to part of the predicted intracytoplasmic domain was used. Strong immunoreactivity was observed on the membrane of most of the epithelial cells examined, including transitional cells of the renal pelvis and ureter, glandular cells of the gastrointestinal tract, renal tubuli, bronchi and pancreas, and stratified epithelium of the oral cavity, trachea and esophagus in this gestational period. A much more intense reaction was observed on the basolateral sides than on the apical side of these cells. No immunoreactivity was found in the liver, adrenal gland, striated and smooth muscles, brain, endothelium or fibroblasts. Western blot analysis confirmed increased expression of the c-erbB-2 gene product in fetal kidney and intestine but not in the brain. As the protein seems to be poorly expressed in normal adult epithelial cells except for renal tubuli, the present results suggest that the protein is a membrane-associated receptor protein which controls some specific reaction of fetal epithelium.

  7. Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

    PubMed Central

    Prinz, W A; Beckwith, J

    1994-01-01

    To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm. PMID:7929016

  8. CONSTRUCTION OF SILKWORM MIDGUT cDNA LIBRARY FOR SCREEN AND SEQUENCE ANALYSIS OF PERITROPHIC MEMBRANE PROTEIN GENES.

    PubMed

    Zhou, Yi-Jun; Xue, Bin; Li, Yang-Yang; Li, Fan-Chi; Ni, Min; Shen, Wei-De; Gu, Zhi-Ya; Li, Bing; Shen, Wei-De; Gu, Zhi-Ya; Li, Bing

    2016-01-01

    Silkworm is an important economic insect and the model species for Lepidoptera. The midgut of silkworm is an important physiological barrier, as its peritrophic membrane (PM) can resist pathogen invasion. In this study, a silkworm midgut cDNA library was constructed in order to identify silkworm PM genes. The capacity of the initial library was 6.92 × 10(6) pfu/ml, along with a recombination rate of 92.14% and a postamplification titer of 4.10 × 10(9) pfu/ml. Three silkworm PM protein genes were obtained by immunoscreening, two of which were chitin-binding protein (CBP) genes and one of which was a chitin deacetylase (CDA) gene as revealed by sequence analysis. Three genes were named BmCBP02, BmCBP13, and BmCDA17, and their ORF sizes are 678, 1,029, and 645 bp, respectively; all of them contain sequences of chitin-binding domains. Phylogenetic analysis indicated that BmCBP02 has the highest consensus with Mamestra configurata CBP at 61.0%; BmCBP13 has the highest consensus with Loxostege sticticalis PM CBP at 53.35%; BmCDA17 has the highest consensus with Helicoverpa armigera CDA5a at 70.83%. Tissue transcriptional analysis revealed that all three genes were specifically expressed in the midgut, and during the developmental process of fifth-instar silkworms, the transcription of all the genes showed an upward trend. This study laid a foundation for further studies on the functions of silkworm PM genes.

  9. Proteins of Excitable Membranes

    PubMed Central

    Nachmansohn, David

    1969-01-01

    Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment. PMID:19873642

  10. Striking Diversity of vmp1, a Variable Gene Encoding a Putative Membrane Protein of the Stolbur Phytoplasma▿

    PubMed Central

    Cimerman, Agnès; Pacifico, Davide; Salar, Pascal; Marzachì, Cristina; Foissac, Xavier

    2009-01-01

    Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the “Candidatus Phytoplasma asteris” genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions. PMID:19270150

  11. Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

    PubMed Central

    Omata, Tatsuo; Carlson, Thomas J.; Ogawa, Teruo; Pierce, John

    1990-01-01

    A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO2-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO2. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO2 conditions. The activity of inorganic carbon (Ci) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO2 was the main species of Ci transported and the activities of CO2 and HCO3− transport increased when high CO2-grown cells were exposed to low CO2. We conclude that the 42-kilodalton protein is not directly involved in the Ci-accumulating mechanism of Synechococcus PCC 7942. Images Figure 3 PMID:16667451

  12. Analysis of Transcriptionally Active Gene Clusters of Major Outer Membrane Protein Multigene Family in Ehrlichia canis and E. chaffeensis

    PubMed Central

    Ohashi, Norio; Rikihisa, Yasuko; Unver, Ahmet

    2001-01-01

    Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis and E. chaffeensis. Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensis genomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis, in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5′-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from −8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3′-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster in E. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5′ and the 3′ sides. Although omp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them. PMID:11254561

  13. An epidemiologic study of mitochondrial membrane transporter protein gene polymorphism and risk factors for neural tube defects in Shanxi, China.

    PubMed

    Liu, Zhizhen; Xie, Jun; Luo, Tian'e; Zhang, Tao; Zhao, Xia; Zhao, Hong; Li, Peizhen

    2012-02-25

    The present study involved a questionnaire survey of 156 mothers that gave birth to children with neural tube defects or had a history of pregnancy resulting in children with neural tube defects (case group) and 156 control mothers with concurrent healthy children (control group) as well as detection of mitochondrial membrane transporter protein gene [uncoupling protein 2 (UCP2)] polymorphism. The maternal UCP2 3' untranslated region (UTR) D/D genotype and D allele frequency were significantly higher in the case group compared with the control group (odds ratio (OR) 3.233; 95% confidence interval (CI) 1.103-9.476; P = 0.040; OR: 3.484; 95% CI: for neural tube defects 2.109-5.753; P < 0.001). Univariate and multivariate logistic regression analysis of risk factors for neural tube defects showed that a maternal UCP2 3' UTR D/D genotype was negatively interacted with the mothers' consumption of frequent fresh fruit and vegetables (S = 0.007), positively interacted with the mothers' frequency of germinated potato consumption (S = 2.15) and positively interacted with the mothers' body mass index (S = 3.50). These findings suggest that maternal UCP2 3' UTR gene polymorphism, pregnancy time, consumption of germinated potatoes and body mass index are associated with an increased risk for neural tube defects in children from mothers living in Shanxi province, China. Moreover, there is an apparent gene-environment interaction involved in the development of neural tube defects in offspring.

  14. Tracking membrane protein association in model membranes.

    PubMed

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a

  15. Membrane fission by protein crowding.

    PubMed

    Snead, Wilton T; Hayden, Carl C; Gadok, Avinash K; Zhao, Chi; Lafer, Eileen M; Rangamani, Padmini; Stachowiak, Jeanne C

    2017-04-18

    Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

  16. Proteins causing membrane fouling in membrane bioreactors.

    PubMed

    Miyoshi, Taro; Nagai, Yuhei; Aizawa, Tomoyasu; Kimura, Katsuki; Watanabe, Yoshimasa

    2015-01-01

    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs.

  17. Cloning, nucleotide sequence, and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein.

    PubMed Central

    Vizcaíno, N; Cloeckaert, A; Zygmunt, M S; Dubray, G

    1996-01-01

    The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective

  18. Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

    PubMed

    Rudtanatip, Tawut; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2015-11-01

    Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Conformation of Membrane Proteins: Bacteriorhodopsin

    DTIC Science & Technology

    1994-05-13

    membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol MINE (MW = 5000) succinimidyl carbonate. The...membrane protein (248 amino acids) which catalyzes the light-induced proton translocation across the membrane of Halobacterium halobium. Research was...purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (MW = 5000) succinimidyl carbonate. The

  20. Calmodulin-binding proteins in bryophytes: identification of abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-bound transporter-like proteins.

    PubMed

    Takezawa, Daisuke; Minami, Anzu

    2004-04-30

    Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca2+ and a Ca(2+)-binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins.

  1. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed

    Yan, X; Gonzales, R A; Wagner, G J

    1997-11-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

  2. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed Central

    Yan, X; Gonzales, R A; Wagner, G J

    1997-01-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM. PMID:9390428

  3. Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa).

    PubMed

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon

    2011-09-01

    Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.

  4. Evaluation of a real-time polymerase chain reaction assay of the outer membrane protein P2 gene for the detection of Haemophilus parasuis in clinical samples

    PubMed Central

    McDowall, Rebeccah; Slavic, Durda; MacInnes, Janet I.; Cai, Hugh Y.

    2014-01-01

    A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively. PMID:24688178

  5. The association of membrane frizzled-related protein (MFRP) gene with acute angle-closure glaucoma – a pilot study

    PubMed Central

    Wang, I-Jong; Lin, Shan; Chiang, Ting-Hsuan; Chen, Zoe Tzu-Yi; Lin, Luke L.K.; Hung, Por-Tying

    2008-01-01

    Purpose The membrane frizzled-related protein (MFRP) has been proposed as a probable candidate gene for extreme hyperopia and nanophthalmos, which are factors for angle-closure glaucoma. The purpose of our study was to investigate whether there are significant associations between angle-closure glaucoma and sequence variants in the MFRP gene reported previously in Taiwanese subjects. Methods Genomic DNA was collected from 63 subjects with angle-closure glaucoma and 66 age-matched and gender-matched controls without angle-closure glaucoma. Three sequence variants were detected by polymerase chain reaction (PCR) and direct sequencing in all of the cases and controls. Results None of the three sequence variants showed a significant result in terms of association with disease. The pairwise linkage disequilibrium (LD) mapping confirmed that these alleles have a comparatively strong LD index greater than 0.7 for D' and greater than 0.4 for r2 at these polymorphisms. However, we found there were no statistical associations between any of the three sequence variants located on MFRP and angle-closure glaucoma. Conclusions In our pilot study, variations that we tested in MFRP were not associated with the development of acute angle-closure glaucoma in Taiwanese subjects. PMID:18781223

  6. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  7. closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition.

    PubMed

    Ventura, Gemma; Furriols, Marc; Martín, Nicolás; Barbosa, Vitor; Casanova, Jordi

    2010-08-01

    The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.

  8. Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

    PubMed Central

    McEwen, J; Silverman, P M

    1982-01-01

    Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins. Images PMID:7050092

  9. Design of membrane proteins: toward functional systems.

    PubMed

    Ghirlanda, Giovanna

    2009-12-01

    Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.

  10. Disease association with two Helicobacter pylori duplicate outer membrane protein genes, homB and homA

    PubMed Central

    Oleastro, Monica; Cordeiro, Rita; Yamaoka, Yoshio; Queiroz, Dulciene; Mégraud, Francis; Monteiro, Lurdes; Ménard, Armelle

    2009-01-01

    Background homB encodes a Helicobacter pylori outer membrane protein. This gene was previously associated with peptic ulcer disease (PUD) and was shown to induce activation of interleukin-8 secretion in vitro, as well as contributing to bacterial adherence. Its 90%-similar gene, homA, was previously correlated with gastritis. The present study aimed to evaluate the gastric disease association with homB and homA, as well as with the H. pylori virulence factors cagA, babA and vacA, in 415 H. pylori strains isolated from patients from East Asian and Western countries. The correlation among these genotypes was also evaluated. Results Both homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains. In Western strains (n = 234, 124 PUD and 110 non-ulcer dyspepsia (NUD), homB, cagA and vacA s1 were all significantly associated with PUD (p = 0.025, p = 0.014, p = 0.039, respectively), and homA was closely correlated with NUD (p = 0.072). In East Asian strains (n = 138, 73 PUD and 65 NUD), homB was found more frequently than homA, and none of these genes was associated with the clinical outcome. Overall, homB was associated with the presence of cagA (p = 0.043) and vacA s1 (p < 0.001), whereas homA was found more frequently in cagA-negative (p = 0.062) and vacA s2 (p < 0.001) strains. Polymorphisms in homB and homA copy number were observed, with a clear geographical specificity, suggesting an involvement of these genes in host adaptation. A correlation between the homB two-copy genotype and PUD was also observed, emphasizing the role of homB in the virulence of the strain. Conclusion The global results suggest that homB and homA contribute to the determination of clinical outcome. PMID:19545429

  11. cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolor A3(2)

    PubMed Central

    2009-01-01

    Background Streptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying differentiation and antibiotic production. To date, many genes have been identified to be required for its differentiation (e.g. bld genes for aerial growth and whi genes for sporulation) and antibiotics production (including actII-orf4, redD, cdaR as pathway-specific regulatory genes and afsR, absA1/A2 as pleiotropic regulatory genes). Results A gene cluster containing six genes (SCO4126-4131) was proved to be co-transcribed in S. coelicolor. Deletions of cmdABCDEF (SCO4126-4131) displayed defective sporulation including formation of aberrant branches, and abnormalities in chromosome segregation and spore septation. Disruption mutants of apparently orthologous genes of S. lividans and S. avermitilis also showed defective sporulation, implying that the role of these genes is similar among Streptomyces. Transcription of cmdB, and therefore presumably of the whole operon, was regulated developmentally. Five of the encoded proteins (CmdA, C, D, E, F) were predicted membrane proteins. The other, CmdB, a predicted ATP/GTP-binding protein with an ABC-transporter-ATPase domain shown here to be essential for its function, was also located on the cell membrane. These results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation at an early stage of aerial hyphae formation, and suggest that these proteins may form a complex on cell membrane for proper segregation of chromosomes. In addition, deletions of cmdABCDEF also revealed over-production of blue-pigmented actinorhodin (Act) via activation of transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis. Conclusion In this study, six co-transcribed genes cmdABCDEF were identified by their effects on differentiation and antibiotic production in Streptomyces coelicolor A3(2). These six membrane-located proteins are possibly assembled into a complex to

  12. Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain.

    PubMed

    Magazin, M D; Moores, J C; Leong, J

    1986-01-15

    Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.

  13. In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

    PubMed Central

    Cartwright, C P; Tipper, D J

    1991-01-01

    Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells. Images PMID:2017168

  14. Epstein-Barr virus gene expression and latent membrane protein 1 gene polymorphism in pediatric liver transplant recipients.

    PubMed

    Kasztelewicz, Beata; Jankowska, Irena; Pawłowska, Joanna; Teisseyre, Joanna; Dzierżanowska-Fangrat, Katarzyna

    2011-12-01

    Immunosuppressed pediatric transplant recipients are at risk of developing Epstein-Barr virus (EBV)-associated complications (such as post-transplant lymphoproliferative disorders). Monitoring of the EBV DNA level in blood alone has a low predictive value for the post-transplant course of EBV infection and its complications. Therefore, additional prognostic markers are widely sought. The study aim was to analyze EBV gene expression patterns and LMP1 polymorphism in relation to EBV DNA levels in pediatric liver transplant recipients. EBV load measurement, LMP1 variant, and gene expression analysis were performed in collected prospectively multiple blood samples from 30 patients. Several distinct patterns of EBV gene expression were identified: latency 2 (71%), latency 3 (13%), latency 0 (11%), and lytic infection (5%). In most children's multiple blood samples, both EBV gene expression patterns and expression levels of individual EBV genes varied significantly over time. EBV gene expression patterns were not associated with the EBV load. However, the viral load correlated with the LMP1 and LMP2 expression (r = 0.34; P  = 0.006, and r  = 0.45; P = 0.001, respectively). Two variants of the LMP1 gene were detected, and they were consistent over time in individual patients. A wild type of LMP1 was associated with higher EBV-DNA loads (P = 0.03). This indicates that EBV infection in immunosuppressed patients is a very dynamic process, but changes in the state of EBV infection do not influence significantly the viral load. The latter, however, can be associated with the activity of LMP1 and LMP2 genes, as well as polymorphism of LMP1.

  15. Protein engineering methods applied to membrane protein targets.

    PubMed

    Lluis, M W; Godfroy, J I; Yin, H

    2013-02-01

    Genes encoding membrane proteins have been estimated to comprise as much as 30% of the human genome. Among these membrane, proteins are a large number of signaling receptors, transporters, ion channels and enzymes that are vital to cellular regulation, metabolism and homeostasis. While many membrane proteins are considered high-priority targets for drug design, there is a dearth of structural and biochemical information on them. This lack of information stems from the inherent insolubility and instability of transmembrane domains, which prevents easy obtainment of high-resolution crystals to specifically study structure-function relationships. In part, this lack of structures has greatly impeded our understanding in the field of membrane proteins. One method that can be used to enhance our understanding is directed evolution, a molecular biology method that mimics natural selection to engineer proteins that have specific phenotypes. It is a powerful technique that has considerable success with globular proteins, notably the engineering of protein therapeutics. With respect to transmembrane protein targets, this tool may be underutilized. Another powerful tool to investigate membrane protein structure-function relationships is computational modeling. This review will discuss these protein engineering methods and their tremendous potential in the study of membrane proteins.

  16. Identification and Comparative Analysis of Genes Encoding Outer Membrane Proteins P2 and P5 in Haemophilus parsuis

    USDA-ARS?s Scientific Manuscript database

    Haemophilus parasuis is a serious swine pathogen but little is known about how it causes disease. A related human pathogen, Haemophilus influenzae, has been better studied and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, have been shown to ha...

  17. Distinct roles of the pepper pathogen-induced membrane protein gene CaPIMP1 in bacterial disease resistance and oomycete disease susceptibility.

    PubMed

    Hong, Jeum Kyu; Choi, Du Seok; Kim, Sang Hee; Yi, Seung Yeon; Kim, Young Jin; Hwang, Byung Kook

    2008-08-01

    Plant integral membrane proteins have essential roles in diverse internal and external physiological processes as signal receptors or ion transporters. The pepper CaPIMP1 gene encoding a putative integral membrane protein with four transmembrane domains was isolated and functionally characterized from pepper leaves infected with the avirulent strain Xanthomonas campestris pv. vesicatoria (Xcv). CaPIMP1-green fluorescence protein (GFP) fusions localized to the plasma membrane in onion cells, as observed by confocal microscopy. CaPIMP1 was expressed in an organ-specific manner in healthy pepper plants. Infection with Xcv induced differential accumulation of CaPIMP1 transcripts in pepper leaf tissues during compatible and incompatible interactions. The function of CaPIMP1 was examined by using the virus-induced gene silencing technique in pepper plants and by overexpression in Arabidopsis. CaPIMP1-silenced pepper plants were highly susceptible to Xcv infection and expressed lower levels of the defense-related gene CaSAR82A. CaPIMP1 overexpression (CaPIMP1-OX) in transgenic Arabidopsis conferred enhanced resistance to P. syringae pv. tomato infection, accompanied by enhanced AtPDF1.2 gene expression. In contrast, CaPIMP1-OX plants were highly susceptible to the biotrophic oomycete Hyaloperonospora parasitica. Taken together, we propose that CaPIMP1 plays distinct roles in both bacterial disease resistance and oomycete disease susceptibility.

  18. A lacZ-pbpB gene fusion coding for an inducible hybrid protein that recognizes localized sites in the inner membrane of Escherichia coli.

    PubMed Central

    Ayala, J A; Plá, J; Desviat, L R; de Pedro, M A

    1988-01-01

    An in-phase gene fusion consisting of the 5'-terminal 1,314 base pairs (bp) of the structural gene for beta-galactosidase (lacZ) and the 3'-terminal 1,644 bp of the structural gene coding for penicillin-binding protein 3 (pbpB) of Escherichia coli was constructed and cloned in the plasmid pDIAM64. The product of the fusion gene was a remarkably stable protein with an apparent molecular weight of 110,000 (p110) that retained the ability to covalently interact with beta-lactam antibiotics. The fusion protein was found associated with the membrane at low levels of induction, but it accumulated in the cytoplasm of cells induced for a long time as inclusion bodies of high density. Inclusion bodies were localized at defined positions corresponding to septal sites in all of the pDIAM64-containing strains tested except PAT84 and GD113 (which carry the ftsZ84 mutant allele). These findings indicate a possible role of the FtsZ protein in the integration of Pbp3 into the membrane and in septum localization during the cell division cycle. Images PMID:3136138

  19. Molecular dynamics of membrane proteins.

    SciTech Connect

    Woolf, Thomas B.; Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  20. Ponticulin is an atypical membrane protein

    PubMed Central

    1994-01-01

    We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha- helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s). PMID:8089175

  1. Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

    PubMed Central

    Haake, D A; Champion, C I; Martinich, C; Shang, E S; Blanco, D R; Miller, J N; Lovett, M A

    1993-01-01

    Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane

  2. Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

    PubMed

    Peters, Swaantje; Tatar, Olcay; Spitzer, Martin S; Szurman, Peter; Aisenbrey, Sabine; Lüke, Matthias; Adam, Annemarie; Yoeruek, Efdal; Grisanti, Salvatore

    2009-02-01

    Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

  3. Structural Symmetry in Membrane Proteins.

    PubMed

    Forrest, Lucy R

    2015-01-01

    Symmetry is a common feature among natural systems, including protein structures. A strong propensity toward symmetric architectures has long been recognized for water-soluble proteins, and this propensity has been rationalized from an evolutionary standpoint. Proteins residing in cellular membranes, however, have traditionally been less amenable to structural studies, and thus the prevalence and significance of symmetry in this important class of molecules is not as well understood. In the past two decades, researchers have made great strides in this area, and these advances have provided exciting insights into the range of architectures adopted by membrane proteins. These structural studies have revealed a similarly strong bias toward symmetric arrangements, which were often unexpected and which occurred despite the restrictions imposed by the membrane environment on the possible symmetry groups. Moreover, membrane proteins disproportionately contain internal structural repeats resulting from duplication and fusion of smaller segments. This article discusses the types and origins of symmetry in membrane proteins and the implications of symmetry for protein function.

  4. Expression of human membrane skeleton protein genes for protein 4.1 and betaIISigma2-spectrin assayed by real-time RT-PCR.

    PubMed

    Taylor-Harris, Pamela M; Felkin, Leanne E; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Barton, Paul J R; Pinder, Jennifer C

    2005-01-01

    The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction.

  5. Phylogenetic profiles of all membrane transport proteins

    PubMed Central

    Weiner, January; Kooij, Taco W.A.

    2016-01-01

    In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date. PMID:28357319

  6. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

    Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

  7. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  8. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  9. The secretory carrier membrane protein family: structure and membrane topology.

    PubMed

    Hubbard, C; Singleton, D; Rauch, M; Jayasinghe, S; Cafiso, D; Castle, D

    2000-09-01

    Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane

  10. Overexpression of a new putative membrane protein gene AtMRB1 results in organ size enlargement in Arabidopsis.

    PubMed

    Guan, Hua; Kang, Dingming; Fan, Min; Chen, Zhangliang; Qu, Li-Jia

    2009-02-01

    Arabidopsis AtMRB1 is predicted to encode a novel protein of 432 amino acid residues in length, with four putative trans-membrane domains. In the present study, characterization of AtMRB1 is conducted. Green fluorescent protein (GFP) fusion protein assay showed that AtMRB1 was located in the plasma membrane. Transgenic lines overexpressing AtMRB1 driven by a CaMV 35S promoter were generated. Statistic analysis showed that, during the seedling stage, the organ sizes of the transgenic lines including hypocotyl length, root length and root weight were significantly larger than those of the wild type plants under both light and dark conditions. In the adult plant stage, the AtMRB1 overexpressor plants were found to have larger organ sizes in terms of leaf length and width, and increased number of cauline leaves and branches when bolting. Further observation indicated that the larger leaf size phenotype was due to a larger number of mesophyll cells, the size of which was not altered. Quantitative real-time polymerase chain reaction analysis showed that the transcription of ANT, ROT3 and GRF5 were upregulated in the AtMRB1-overexpressor plants. These data suggest that AtMRB1 is possibly a positive regulator of organ size development in Arabidopsis, mainly through cell number control.

  11. Thermodynamic competition between membrane protein oligomeric states

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Haselwandter, Christoph A.

    2016-10-01

    Self-assembly of protein monomers into distinct membrane protein oligomers provides a general mechanism for diversity in the molecular architectures, and resulting biological functions, of membrane proteins. We develop a general physical framework describing the thermodynamic competition between different oligomeric states of membrane proteins. Using the mechanosensitive channel of large conductance as a model system, we show how the dominant oligomeric states of membrane proteins emerge from the interplay of protein concentration in the cell membrane, protein-induced lipid bilayer deformations, and direct monomer-monomer interactions. Our results suggest general physical mechanisms and principles underlying regulation of protein function via control of membrane protein oligomeric state.

  12. The gene encoding the GPI-anchored membrane protein p137{sup GPI} (M11S1) maps to human chromosome 11p13 and is highly conserved in the mouse

    SciTech Connect

    Gessler, M.; Klamt, B.; Tsaoussidou, S.

    1996-02-15

    This article reports on the mapping of the gene encoding the GPI-anchored membrane protein p137{sup GPI} (M11S1) to human chromosome 11p13. Genomic clones will help to discern the structure-activity relationships of the gene encoding this protein. 6 refs., 1 fig.

  13. SNARE proteins and 'membrane rafts'.

    PubMed

    Lang, Thorsten

    2007-12-15

    The original 'lipid raft' hypothesis proposed that lipid-platforms/rafts form in the exoplasmic plasmalemmal leaflet by tight clustering of sphingolipids and cholesterol. Their physical state, presumably similar to liquid-ordered phases in model membranes, would confer detergent resistance to rafts and enriched proteins therein. Based on this concept, detergent resistant membranes (DRMs) from solubilized cells were considered to reflect pre-existing 'lipid rafts' in live cells. To date, more than 200 proteins were found in DRMs including also members of the SNARE superfamily, which are small membrane proteins involved in intracellular fusion steps. Their raft association indicates that they are not uniformly distributed, and, indeed, microscopic studies revealed that SNAREs concentrate in submicrometre-sized, cholesterol-dependent clusters at which vesicles fuse. However, the idea that SNARE clusters are 'lipid rafts' was challenged, as they do not colocalize with raft markers, and SNAREs are excluded from liquid-ordered phases in model membranes. Independent from this disagreement, in recent years the solubilization criterion has been criticized for several reasons, calling for a more exact definition of rafts. At a recent consensus on a revised raft model, the term 'lipid rafts' was replaced by 'membrane rafts' that were defined as 'small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes'. As a result, after dismissing the terms 'detergent resistant' and 'liquid-ordered', it now appears that SNARE clusters are bona fide 'membrane rafts'.

  14. Rare mutations and potentially damaging missense variants in genes encoding fibrillar collagens and proteins involved in their production are candidates for risk for preterm premature rupture of membranes

    PubMed Central

    Teves, Maria E.; Pearson, Laurel N.; Parikh, Hardik I.; Chaemsaithong, Piya; Sheth, Nihar U.; York, Timothy P.; Romero, Roberto; Strauss, Jerome F.

    2017-01-01

    Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest—fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans. PMID:28346524

  15. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

    PubMed Central

    Vialard, J; Lalumière, M; Vernet, T; Briedis, D; Alkhatib, G; Henning, D; Levin, D; Richardson, C

    1990-01-01

    An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified

  16. Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca(2+)-ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation.

    PubMed

    Adebayo, Olusegun L; Khera, Alka; Sandhir, Rajat; Adenuga, Gbenga A

    2016-03-01

    The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Strategies for the purification of membrane proteins.

    PubMed

    Smith, Sinead Marian

    2011-01-01

    Although membrane proteins account for 20-30% of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes proteins with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilisation, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction requires phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilisation, where problems are most frequently encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilisation of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of later purification techniques.

  18. Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

    PubMed Central

    Litwin, Christine M.; Byrne, Burke L.

    1998-01-01

    Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

  19. The Schizosaccharomyces pombe spo3+ Gene Is Required for Assembly of the Forespore Membrane and Genetically Interacts with psy1+-encoding Syntaxin-like Protein

    PubMed Central

    Nakamura, Taro; Nakamura-Kubo, Michiko; Hirata, Aiko; Shimoda, Chikashi

    2001-01-01

    Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane. spo3+ encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles, spo3-KC51, was dose-dependently suppressed by psy1+, which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1+ was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In the spo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immature spo3Δ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein. PMID:11739793

  20. Specific detection of Xanthomonas oryzae pv. oryzicola in infected rice plant by use of PCR assay targeting a membrane fusion protein gene.

    PubMed

    Kang, Man Jung; Shim, Jae Kyung; Cho, Min Seok; Seol, Young Joo; Hahn, Jang Ho; Hwang, Duk Ju; Park, Dong Suk

    2008-09-01

    Successful control of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, requires a specific and reliable diagnostic tool. A pathovar-specific PCR assay was developed for the rapid and accurate detection of the plant pathogenic bacterium Xanthomonas oryzae pv. oryzicola in diseased plant. Based on differences in a membrane fusion protein gene of Xanthomonas oryzae pv. oryzicola and other microorganisms, which was generated from NCBI (http://www.ncbi.nlm.nih.gov/) and CMR (http://cmr.tigr.org/) BLAST searches, one pair of pathovar-specific primers, XOCMF/XOCMR, was synthesized. Primers XOCMF and XOCMR from a membrane fusion protein gene were used to amplify a 488-bp DNA fragment. The PCR product was only produced from 4 isolates of Xanthomonas oryzae pv. oryzicola among 37 isolates of other pathovars and species of Xanthomonas, Pectobacterium, Pseudomonas, Burkholderia, Escherichia coli, and Fusarium oxysporum f.sp. dianthi. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods.

  1. Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer membrane protein gene.

    PubMed Central

    Watson, M W; Lambden, P R; Clarke, I N

    1991-01-01

    The 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection. Images PMID:1864938

  2. A Novel Deletion Mutation of Exon 2 of the C19orf12 Gene in an Omani Family with Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN)

    PubMed Central

    Al Macki, Nabil; Al Rashdi, Ismail

    2017-01-01

    Mutations in the C19orf12 gene are known to cause mitochondrial membrane protein-associated neurodegeneration (MPAN), which is a neurodegeneration with brain iron accumulation (NBIA) type 4 disorder. To the best of our knowledge, this is the first report of a genetically confirmed case of MPAN from Oman. A novel homozygous deletion of exon 2 of the C19orf12 gene was confirmed on the proband, a seven-year-old girl, who presented with gait instability. Brain magnetic resonance imaging showed iron deposition on the basal ganglia. This report highlights the importance of genetic testing of such a clinically and genetically heterogeneous condition among a population with a high consanguinity rate. To overcome the diagnostic difficulty, implementation of a cost-effective approach to perform cascade screening of carriers at risk is needed as well as programs to address risky consanguineous marriages. PMID:28042406

  3. Gene Therapy in Patient-specific Stem Cell Lines and a Preclinical Model of Retinitis Pigmentosa With Membrane Frizzled-related Protein Defects

    PubMed Central

    Li, Yao; Wu, Wen-Hsuan; Hsu, Chun-Wei; Nguyen, Huy V; Tsai, Yi-Ting; Chan, Lawrence; Nagasaki, Takayuki; Maumenee, Irene H; Yannuzzi, Lawrence A; Hoang, Quan V; Hua, Haiqing; Egli, Dieter; Tsang, Stephen H

    2014-01-01

    Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrprd6/Mfrprd6 mice—an established preclinical model of RP—and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrprd6/Mfrprd6 preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials. PMID:24895994

  4. Gene therapy in patient-specific stem cell lines and a preclinical model of retinitis pigmentosa with membrane frizzled-related protein defects.

    PubMed

    Li, Yao; Wu, Wen-Hsuan; Hsu, Chun-Wei; Nguyen, Huy V; Tsai, Yi-Ting; Chan, Lawrence; Nagasaki, Takayuki; Maumenee, Irene H; Yannuzzi, Lawrence A; Hoang, Quan V; Hua, Haiqing; Egli, Dieter; Tsang, Stephen H

    2014-09-01

    Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice--an established preclinical model of RP--and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.

  5. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

  6. Membrane stiffness is modified by integral membrane proteins.

    PubMed

    Fowler, Philip W; Hélie, Jean; Duncan, Anna; Chavent, Matthieu; Koldsø, Heidi; Sansom, Mark S P

    2016-09-20

    The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, Kc). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.

  7. Biopores/membrane proteins in synthetic polymer membranes.

    PubMed

    Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

    2017-04-01

    Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

  8. Computational modeling of membrane proteins

    PubMed Central

    Leman, Julia Koehler; Ulmschneider, Martin B.; Gray, Jeffrey J.

    2014-01-01

    The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. PMID:25355688

  9. Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.

    PubMed

    Treves, S; Feriotto, G; Moccagatta, L; Gambari, R; Zorzato, F

    2000-12-15

    Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural

  10. Membrane shape modulates transmembrane protein distribution

    PubMed Central

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E. S.; Bassereau, Patricia

    2014-01-01

    Summary Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown due to the numerous sorting mechanisms taking place concurrently in cells. To isolate the effect of membrane shape, cellsized Giant Unilamellar Vesicles (GUVs) containing either the potassium channel, KvAP, or water channel, AQP0, were used to form membrane nanotubes with controlled radii. While the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. FRAP measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins, and provides a method for determining the effective shape and flexibility of membrane proteins. PMID:24480645

  11. Molecular cloning of cDNAs encoding human GLEPP1, a membrane protein tyrosine phosphatase: characterization of the GLEPP1 protein distribution in human kidney and assignment of the GLEPP1 gene to human chromosome 12p12-p13.

    PubMed

    Wiggins, R C; Wiggins, J E; Goyal, M; Wharram, B L; Thomas, P E

    1995-05-01

    Human glomerular epithelial protein 1 (GLEPP1), a receptor-like membrane protein tyrosine phosphatase (PTPase), was cloned and sequenced from a human renal cortical cDNA library. The human nucleotide and derived amino acid sequences were, respectively, 90 and 97% identical to those of rabbit. Human GLEPP1 is predicted to contain 1188 amino acids. The predicted mature protein is 1159 amino acids long and contains a large extracellular domain, a single transmembrane domain, and a single intracellular PTPase domain. Monoclonal and polyclonal antibodies raised against a human GLEPP1 fusion protein recognized a protein with distribution restricted to the glomerulus in human kidney and with an apparent molecular weight of approximately 200 kDa. The GLEPP1 gene was assigned to human chromosome 12p12-p13 by fluorescence in situ hybridization.

  12. Membrane Structure: Lipid-Protein Interactions in Microsomal Membranes*

    PubMed Central

    Trump, Benjamin F.; Duttera, Sue M.; Byrne, William L.; Arstila, Antti U.

    1970-01-01

    The relationships of phospholipid to membrane structure and function were examined in hepatic microsomes. Findings indicate that normal microsomal membrane structure is dependent on lipid-protein interactions and that it correlates closely with glucose-6-phosphatase activity. Modification of most phospholipid with phospholipase-C is associated with widening of the membrane which can be reversed following readdition of phospholipid. Images PMID:4317915

  13. Membrane proteins: always an insoluble problem?

    PubMed Central

    Rawlings, Andrea E.

    2016-01-01

    Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence. PMID:27284043

  14. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes.

    PubMed

    Magrì, Andrea; Di Rosa, Maria Carmela; Tomasello, Marianna Flora; Guarino, Francesca; Reina, Simona; Messina, Angela; De Pinto, Vito

    2016-06-01

    Cu/Zn Superoxide Dismutase (SOD1), the most important antioxidant defense against ROS in eukaryotic cells, localizes in cytosol and intermembrane space of mitochondria (IMS). Several evidences show a SOD1 intersection with both fermentative and respiratory metabolism. The Voltage Dependent Anion Channel (VDAC) is the main pore-forming protein in the mitochondrial outer membrane (MOM), and is considered the gatekeeper of mitochondrial metabolism. Saccharomyces cerevisiae lacking VDAC1 (Δpor1) is a very convenient model system, since it shows an impaired growth rate on non-fermentable carbon source. Transformation of Δpor1 yeast with human SOD1 completely restores the cell growth deficit in non-fermentative conditions and re-establishes the physiological levels of ROS, as well as the mitochondrial membrane potential. No similar result was found upon yeast SOD1 overexpression. A previous report highlighted the action of SOD1 as a transcription factor. Quantitative Real-Time PCR showed that β-barrel outer-membrane encoding-genes por2, tom40, sam50 are induced by hSOD1, but the same effect was not obtained in Δpor1Δpor2 yeast, indicating a crucial function for yVDAC2. Since the lack of VDAC1 in yeast can be considered a stress factor for the cell, hSOD1 could relieve it stimulating the expression of genes bringing to the recovery of the MOM function. Our results suggest a direct influence of SOD1 on VDAC.

  15. Membrane tension and peripheral protein density mediate membrane shape transitions

    NASA Astrophysics Data System (ADS)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

  16. Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

    PubMed Central

    Cloeckaert, A; Verger, J M; Grayon, M; Zygmunt, M S; Grépinet, O

    1996-01-01

    The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar

  17. Detection of Proteins on Blot Membranes.

    PubMed

    Goldman, Aaron; Harper, Sandra; Speicher, David W

    2016-11-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc.

  18. Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Hoehn, G T; Clark, V L

    1992-01-01

    When grown under anaerobic conditions, Neisseria gonorrhoeae, the etiologic agent of the sexually transmitted disease gonorrhea, expresses several novel outer membrane proteins. One of these, Pan 1, has an apparent molecular mass of 54 kDa in electrophoresis and is recognized by serum samples from patients with gonococcal infection. The presence of antibodies to this protein in patient sera suggests that Pan 1 is expressed during gonococcal infection and, more importantly, that N. gonorrhoeae grows anaerobically in vivo. We have cloned the Pan 1 structural gene, aniA, by screening a gonococcal lambda gt11 expression library with monospecific, polyclonal anti-Pan 1 antiserum. Three distinct immunoreactive recombinants, containing overlapping fragments of DNA, were isolated and confirmed to be coding for Pan 1 protein sequences. Northern (RNA blot) hybridization of an insert from an aniA recombinant to total gonococcal cellular RNA revealed the presence of a 1.5-kb transcript that was specific to RNA from anaerobically grown gonococci, indicating that the aniA gene is regulated at the transcriptional level and is monocistronic. To characterize the aniA gene, we have sequenced the entire 2-kb region spanned by the overlapping recombinants. We have also performed primer extension analysis on RNA isolated from aerobically and anaerobically grown gonococci in order to define the aniA promoter region. Two putative primer extension products specific to organisms grown anaerobically were identified by homology to known Escherichia coli promoter sequences, suggesting that the regulation of aniA expression involves multiple promoter regions. Images PMID:1383156

  19. Malate synthase a membrane protein

    SciTech Connect

    Chapman, K.D.; Turley, R.B.; Hermerath, C.A.; Carrapico, F.; Trelease, R.N.

    1987-04-01

    Malate synthase (MS) is generally regarded as a peripheral membrane protein, and believed by some to be ontogenetically associated with ER. However, immuno- and cyto-chemical in situ localizations show MS throughout the matrix of cotton (and cucumber) glyoxysomes, not specifically near their boundary membranes, nor in ER. Only a maximum of 50% MS can be solubilized from cotton glyoxysomes with 1% Triton X-100, 2mM Zwittergen 14, or 10mM DOC +/- salts. Cotton MS does not incorporate /sup 3/H-glucosamine in vivo, nor does it react with Con A on columns or blots. Cotton MS banded with ER in sucrose gradients (20-40%) in Tricine after 3h, but not after 22h in Tricine or Hepes, or after 3h in Hepes or K-phosphate. Collectively the authors data are inconsistent with physiologically meaningful MS-membrane associations in ER or glyoxysomes. It appears that experimentally-induced aggregates of MS migrate in ER gradients and occur in isolated glyoxysomes. These data indicate that ER is not involved in synthesis or modification of cottonseed MS prior to its import into the glyoxysomal matrix.

  20. Protein-Induced Membrane Curvature Alters Local Membrane Tension

    PubMed Central

    Rangamani, Padmini; Mandadap, Kranthi K.; Oster, George

    2014-01-01

    Adsorption of proteins onto membranes can alter the local membrane curvature. This phenomenon has been observed in biological processes such as endocytosis, tubulation, and vesiculation. However, it is not clear how the local surface properties of the membrane, such as membrane tension, change in response to protein adsorption. In this article, we show that the partial differential equations arising from classical elastic model of lipid membranes, which account for simultaneous changes in shape and membrane tension due to protein adsorption in a local region, cannot be solved for nonaxisymmetric geometries using straightforward numerical techniques; instead, a viscous-elastic formulation is necessary to fully describe the system. Therefore, we develop a viscous-elastic model for inhomogeneous membranes of the Helfrich type. Using the newly available viscous-elastic model, we find that the lipids flow to accommodate changes in membrane curvature during protein adsorption. We show that, at the end of protein adsorption process, the system sustains a residual local tension to balance the difference between the actual mean curvature and the imposed spontaneous curvature. We also show that this change in membrane tension can have a functional impact such as altered response to pulling forces in the presence of proteins. PMID:25099814

  1. Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis.

    PubMed

    Ben Romdhane, Walid; Ben-Saad, Rania; Meynard, Donaldo; Verdeil, Jean-Luc; Azaza, Jalel; Zouari, Nabil; Fki, Lotfi; Guiderdoni, Emmanuel; Al-Doss, Abdullah; Hassairi, Afif

    2017-03-24

    We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H₂O₂, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na⁺ but higher K⁺ and Ca(2+) than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

  2. Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis

    PubMed Central

    Ben Romdhane, Walid; Ben-Saad, Rania; Meynard, Donaldo; Verdeil, Jean-Luc; Azaza, Jalel; Zouari, Nabil; Fki, Lotfi; Guiderdoni, Emmanuel; Al-Doss, Abdullah; Hassairi, Afif

    2017-01-01

    We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H2O2, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na+ but higher K+ and Ca2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants. PMID:28338609

  3. Internal packing of helical membrane proteins

    PubMed Central

    Eilers, Markus; Shekar, Srinivasan C.; Shieh, Ted; Smith, Steven O.; Fleming, Patrick J.

    2000-01-01

    Helix packing is important in the folding, stability, and association of membrane proteins. Packing analysis of the helical portions of 7 integral membrane proteins and 37 soluble proteins show that the helices in membrane proteins have higher packing values (0.431) than in soluble proteins (0.405). The highest packing values in integral membrane proteins originate from small hydrophobic (G and A) and small hydroxyl-containing (S and T) amino acids, whereas in soluble proteins large hydrophobic and aromatic residues have the highest packing values. The highest packing values for membrane proteins are found in the transmembrane helix–helix interfaces. Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins. These observations are consistent with a shorter axial separation between helices in membrane proteins. The tight helix packing revealed in this analysis contributes to membrane protein stability and likely compensates for the lack of the hydrophobic effect as a driving force for helix–helix association in membranes. PMID:10823938

  4. A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators.

    PubMed Central

    Ueda, K; Miyake, K; Horinouchi, S; Beppu, T

    1993-01-01

    Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1. Images PMID:8458843

  5. Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

    PubMed Central

    Calcagno, Anna Maria; Chewning, Katherine J; Wu, Chung-Pu; Ambudkar, Suresh V

    2006-01-01

    Background Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. Results In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. Conclusion We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors. PMID:16978418

  6. Artificial membranes for membrane protein purification, functionality and structure studies.

    PubMed

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    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. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  7. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

    PubMed

    Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

    2004-07-01

    Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

  8. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  9. Mycoplasma gallisepticum Lipid Associated Membrane Proteins Up-regulate Inflammatory Genes in Chicken Tracheal Epithelial Cells via TLR-2 Ligation through an NF-κB Dependent Pathway

    PubMed Central

    Majumder, Sanjukta; Zappulla, Frank; Silbart, Lawrence K.

    2014-01-01

    Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (Rlow) or a non-virulent (Rhigh) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, Rlow exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2–3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either Rlow or Rhigh exposure. Taken together we conclude that LAMPs isolated from both Rhigh and Rlow induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway. PMID:25401327

  10. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  11. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  12. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  13. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

    The plasma membrane (PM) and endocytic protein quality control (QC) in conjunction with the endosomal sorting machinery either repairs or targets conformationally damaged membrane proteins for lysosomal/vacuolar degradation. Here, we provide an overview of emerging aspects of the underlying mechanisms of PM QC that fulfill a critical role in preserving cellular protein homeostasis in health and diseases. PMID:24985330

  14. Tuning microbial hosts for membrane protein production

    PubMed Central

    2009-01-01

    The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months. This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production. PMID:20040113

  15. Silencing of the Epstein-Barr Virus Latent Membrane Protein 1 Gene by the Max-Mad1-mSin3A Modulator of Chromatin Structure

    PubMed Central

    Sjöblom-Hallén, Anna; Yang, Weiwen; Jansson, Ann; Rymo, Lars

    1999-01-01

    The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells. PMID:10074148

  16. Rapid Method for Species-Specific Identification of Vibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW

    PubMed Central

    Nandi, Bisweswar; Nandy, Ranjan K.; Mukhopadhyay, Sarmishtha; Nair, G. Balakrish; Shimada, Toshio; Ghose, Asoke C.

    2000-01-01

    The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (∼98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW- and toxR-specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one (V. mimicus) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus, V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin. PMID:11060082

  17. Protein quality control at the inner nuclear membrane.

    PubMed

    Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O; Knop, Michael

    2014-12-18

    The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

  18. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  19. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life

    PubMed Central

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-01-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. PMID:27501943

  20. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    PubMed

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  1. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    PubMed Central

    Milano, Teresa

    2016-01-01

    The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups. PMID:27446613

  2. Regulation of the beta-lactamase BlaL of Streptomyces cacaoi: the product of the blaB regulatory gene is an internal membrane-bound protein.

    PubMed Central

    Magdalena, J; Joris, B; Van Beeumen, J; Brasseur, R; Dusart, J

    1995-01-01

    The beta-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by beta-lactam compounds. This regulation has been shown to depend on the products of two open reading frames, ORF1 (blaA) and ORF2 (blaB) [Lenzini, Magdalena, Fraipont, Joris, Matagne and Dusart (1992) Mol. Gen. Genet. 235, 41-48]. BlaA belongs to the LysR family of transcription activators, whereas BlaB shares some features with the penicillin-recognizing proteins. BlaB has now been overexpressed in Escherichia coli, purified and used for antibody preparation. Immunoblotting of cell-fractionated materials from S. cacaoi showed that BlaB is attached to the internal face of the cytoplasmic membrane. It could not be released by high salt concentrations or EDTA, but only by protease treatment. Under the assay conditions, BlaB did not act as a penicillin-binding protein, a beta-lactamase, a D-amino-peptidase or a target in a phosphorylation step. Images Figure 2 Figure 3 Figure 4 PMID:7575447

  3. Comparative molecular biological analysis of membrane transport genes in organisms

    PubMed Central

    Nagata, Toshifumi; Iizumi, Shigemi; Satoh, Kouji

    2008-01-01

    Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport. Electronic supplementary material The online version of this article (doi:10.1007/s11103-007-9287-z) contains supplementary material, which is available to authorized users. PMID:18293089

  4. Metagenomic Identification of a Novel Salt Tolerance Gene from the Human Gut Microbiome Which Encodes a Membrane Protein with Homology to a brp/blh-Family β-Carotene 15,15′-Monooxygenase

    PubMed Central

    Culligan, Eamonn P.; Sleator, Roy D.; Marchesi, Julian R.; Hill, Colin

    2014-01-01

    The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane. PMID:25058308

  5. [Membrane protein characterization by photoactivatable localization microscopy].

    PubMed

    Huang, Li; Fang, Weihuan; Yu, Ying; Song, Houhui

    2012-11-01

    The on-site labeling and localization tracking of membrane proteins in pathogenic bacteria are tedious work. In order to develop a novel protein labeling technology at super resolution level (nanometer scale) using the photoactivatable localization microscopy (PALM), the chimeric protein of the outer membrane protein A (OmpA) of Mycobacterium tuberculosis and the photoactivatable mEos2m protein were expressed in the non-pathogenic Mycobacterium smegmatis. The recombinant bacteria were fixed on slide, activated by 405 nm laser and subject to PALM imaging to capture photons released by the fusion protein. Meanwhile, colony and cell morphology were visualized under regular fluorescent stereomicroscope and upright fluorescent microscope to characterize fluorescence conversion and protein localization. The fusion proteins formed a "belt"-like structure on cell membrane of M. smegmatis under PALM, providing direct evidence of on-site imaging of membrane proteins. Expression of fusion protein did not compromise the localization properties of OmpA. Thus, mEos2m could be used as a labeling probe to track localizations of non-oligomer oriented membrane proteins. This indicates non-pathogenic M. smegmatis could be served as a model strain to characterize the function and localization of the proteins derived from pathogenic M. tuberculosis. This is the first report using PALM to characterize localization of membrane proteins.

  6. Membrane topology of transmembrane proteins: determinants and experimental tools.

    PubMed

    Lee, Hunsang; Kim, Hyun

    2014-10-17

    Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.

  7. Outer Membrane Protein C (ompC) Gene as the Target for Diagnosis of Salmonella Species Isolated from Human and Animal Sources

    PubMed Central

    Jawad, Alaa Abdel-Kadhim; Al-Charrakh, Alaa H.

    2016-01-01

    Background: The use of selective and differential plating media is a simple method for the isolation of Salmonella spp. Recently, there has been a general move toward molecular methods of Salmonella detection and typing. Methods: A total of 1200 different specimens collected from human and animal sources were involved in his study. 600 stool specimens from patients suffering from diarrhea and 600 specimens from gall bladder (bile) of cattle from Al-Diwaniya slaughter house, Iraq were used. Salmonella spp. were isolated and identified using bacterial culturing on selective media and colonies were tested by API 20Eand then serotyping through polyvalent antisera and conformation by Polymerase Chain Reaction (PCR). PCR was used to detect ompC gene encoding biosynthesis of outer membrane protein C of Salmonella genus. Results: The results revealed that the rate of Salmonella isolates was 0.5% (3/600) from human and 1% (6/600) from animals. The PCR technique revealed that 9 isolates of Salmonella spp. harbored ompC gene. The results of this study revealed that the PCR technique had a high specificity in detection of Salmonella spp., in comparison to culture and biochemical test, Mini API 20 E and serological tests. The present study found no significant differences between human and animal isolates. Conclusion: Detection of ompC gene is a good method for detection of Salmonella species isolated from clinical specimens. It has a high specificity in comparison with other tests, with its advantages of greater speed and effectiveness than conventional detection methods. PMID:26855735

  8. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  9. Membrane Protein Structure Determination in Membrana

    PubMed Central

    DING, YI; YAO, YONG; MARASSI, FRANCESCA M.

    2014-01-01

    CONSPECTUS The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three

  10. Membrane protein structure determination in membrana.

    PubMed

    Ding, Yi; Yao, Yong; Marassi, Francesca M

    2013-09-17

    The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure

  11. Proteomic analysis of protein adsorption capacity of different haemodialysis membranes.

    PubMed

    Urbani, Andrea; Lupisella, Santina; Sirolli, Vittorio; Bucci, Sonia; Amoroso, Luigi; Pavone, Barbara; Pieroni, Luisa; Sacchetta, Paolo; Bonomini, Mario

    2012-04-01

    Protein-adsorptive properties are a key feature of membranes used for haemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with chemical feature of the membrane surfaces. Dialyzers composed of either cellulose triacetate (Sureflux 50 L, effective surface area 0.5 m(2); Nipro Corporation, Japan) or the polysulfone-based helixone (FX40, effective surface area 0.4 m(2); Fresenius Medical Care AG, Germany) materials were employed to develop an ex vivo apparatus to study protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by a proteomic approach. The profiling strategy was based on 2D-electrophoresis separation of desorbed protein coupled to MALDI-TOF/TOF analysis. The total protein adsorption was not significantly different between the two materials. An average of 179 protein spots was visualised for helixone membranes while a map of retained proteins of cellulose triacetate membranes was made up of 239 protein spots. The cellulose triacetate material showed a higher binding capacity for albumin and apolipoprotein. In fact, a number of different protein spots belonging to the gene transcript of albumin were visible in the cellulose triacetate map. In contrast, helixone bound only a small proportion of albumin, while proved to be particularly active in retaining protein associated with the coagulation cascade, such as the fibrinogen isoforms. Our data indicate that proteomic techniques are a useful approach for the investigation of proteins surface-adsorbed onto haemodialysis membranes, and may provide a molecular base for the interpretation of the efficacy and safety of anticoagulation treatment during renal replacement therapy.

  12. Protein Solvation in Membranes and at Water-Membrane Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

    2002-01-01

    Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

  13. Comprehensive Proteomic Analysis of Membrane Proteins in Toxoplasma gondii*

    PubMed Central

    Che, Fa-Yun; Madrid-Aliste, Carlos; Burd, Berta; Zhang, Hongshan; Nieves, Edward; Kim, Kami; Fiser, Andras; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer “sandwich” gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies. PMID:20935347

  14. Crystallization of Membrane protein under Microgravity

    NASA Astrophysics Data System (ADS)

    Henning, C.; Frank, J.; Laubender, G.; Fromme, P.

    2002-01-01

    Proteins are biological molecules which catalyse all essential reactions of cells. The knowledge on the structure of these molecular machines is necessary for the understanding of their function. Many diseases are caused by defects of membrane proteins. In order to develop new medical therapies the construction principle of the proteins must be known. The main difficulty in the determination of the structure of these membrane protein complexes is the crystallisation. Membrane proteins are normally not soluble in water and have therefore to be solubilised from the membranes by use of detergents. The whole protein-detergent micelle must be crystallised to maintain the functional integrity of the protein complexes. These difficulties are the reasons for the fact that crystals of membrane proteins are difficult to grow and most of them are badly ordered, being not appropriate for X-ray structure analysis. The crystallisation of proteins under microgravity leads to the growth of better-ordered crystals by reduction of nucleation rate and the undisturbed growth of the hovering seeds by the absence of sedimentation and convection. The successful crystallistation of a membrane protein under microgravity has been performed during the space shuttle missions USML2 and STS95 in the Space Shuttle with Photosystem I as model protein. Photosystem I is a large membrane protein complex which catalyses one of the first and fundamental steps in oxygen photosynthesis. The crystals of Photosystem I, grown under microgravity were twenty times larger than all Photosystem I crystals which have been grown on earth. They were the basis for the determination of an improved X-ray structure of Photo- system I. These experiments opened the way for the structure enlightenment of more membrane proteins on the basis of microgravity experiments. On board of the International Space Station ideal conditions for the crystallisation of proteins under zero gravity are existing.

  15. IFITM Proteins Restrict Viral Membrane Hemifusion

    PubMed Central

    Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

    2013-01-01

    The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous

  16. A predictor of membrane class: Discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins.

    PubMed

    Taylor, Paul D; Toseland, Christopher P; Attwood, Teresa K; Flower, Darren R

    2006-10-07

    Accurate protein structure prediction remains an active objective of research in bioinformatics. Membrane proteins comprise approximately 20% of most genomes. They are, however, poorly tractable targets of experimental structure determination. Their analysis using bioinformatics thus makes an important contribution to their on-going study. Using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we have addressed the alignment-free discrimination of membrane from non-membrane proteins. The method successfully identifies prokaryotic and eukaryotic alpha-helical membrane proteins at 94.4% accuracy, beta-barrel proteins at 72.4% accuracy, and distinguishes assorted non-membranous proteins with 85.9% accuracy. The method here is an important potential advance in the computational analysis of membrane protein structure. It represents a useful tool for the characterisation of membrane proteins with a wide variety of potential applications.

  17. Membrane Protein Crystallization Using Laser Irradiation

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

    2004-10-01

    We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

  18. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  19. Lateral proton transfer between the membrane and a membrane protein.

    PubMed

    Ojemyr, Linda; Sandén, Tor; Widengren, Jerker; Brzezinski, Peter

    2009-03-17

    Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (approximately 7 x 1010 s(-1) M(-1), compared to the expected value of (1-2) x 10(10) s(-1) M(-1)), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to approximately 3 x 10(13) s(-1) M(-1), which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

  20. Inherently tunable electrostatic assembly of membrane proteins.

    PubMed

    Liang, Hongjun; Whited, Gregg; Nguyen, Chi; Okerlund, Adam; Stucky, Galen D

    2008-01-01

    Membrane proteins are a class of nanoscopic entities that control the matter, energy, and information transport across cellular boundaries. Electrostatic interactions are shown to direct the rapid co-assembly of proteorhodopsin (PR) and lipids into long-range crystalline arrays. The roles of inherent charge variations on lipid membranes and PR variants with different compositions are examined by tuning recombinant PR variants with different extramembrane domain sizes and charged amino acid substitutions, lipid membrane compositions, and lipid-to-PR stoichiometric ratios. Rational control of this predominantly electrostatic assembly for PR crystallization is demonstrated, and the same principles should be applicable to the assembly and crystallization of other integral membrane proteins.

  1. Membrane injury by pore-forming proteins.

    PubMed

    Bischofberger, Mirko; Gonzalez, Manuel R; van der Goot, F Gisou

    2009-08-01

    The plasma membrane defines the boundary of every living cell, and its integrity is essential for life. The plasma membrane may, however, be challenged by mechanical stress or pore-forming proteins produced by the organism itself or invading pathogens. We will here review recent findings about pore-forming proteins from different organisms, highlighting their structural and functional similarities, and describe the mechanisms that lead to membrane repair, since remarkably, cells can repair breaches in their plasma membrane of up to 10,000 microm(2).

  2. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  3. Identification of a Spliced Gene from Kaposi’s Sarcoma-Associated Herpesvirus Encoding a Protein with Similarities to Latent Membrane Proteins 1 and 2A of Epstein-Barr Virus

    PubMed Central

    Glenn, Mark; Rainbow, Lucille; Auradé, Frédéric; Davison, Andrew; Schulz, Thomas F.

    1999-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a novel herpesvirus implicated as the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma, and some cases of multicentric Castleman’s disease. KSHV persists in the majority of KS spindle (endothelial tumor) cells and lymphoid cells in a latent form, with only a limited set of viral genes expressed in a tissue-specific manner. Here, we report the identification of a family of alternatively-spliced transcripts of approximately 7.5 kb expressed in latently infected body cavity-based lymphoma (BCBL) cell lines which are predicted to encode membrane proteins with similarities to the LMP2A and LMP1 proteins of Epstein-Barr virus. In two highly divergent sequence variants of the right end of the KSHV genome, alternative splicing of eight exons located between KSHV ORF 75 and the terminal repeats yields transcripts appropriate for proteins with up to 12 transmembrane domains, followed by a hydrophilic C-terminal, presumably cytoplasmic, domain. This C-terminal domain contains several YxxI/L motifs reminiscent of LMP2A and a putative TRAF binding site as in LMP1. In latently (persistently) infected BCBL cells the predominant transcript utilizes all eight exons, whereas in phorbol-ester-induced cells, a shorter transcript, lacking exons 4 and 5, is also abundant. We also found evidence for an alternative use of exon 1. Transfection of an epitope-tagged cDNA construct containing all exons indicates that the encoded protein is localized on cell surface and intracellular membranes, and glutathione S-transferase pull-down experiments indicate that its cytoplasmic domain, like that of LMP1, interacts with TRAF1, -2, and -3. Two of 20 KS patients had antibodies to the hydrophilic C-terminal domain, suggesting that the protein is expressed in vivo. PMID:10400794

  4. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.

    PubMed

    Knowles, Timothy J; Scott-Tucker, Anthony; Overduin, Michael; Henderson, Ian R

    2009-03-01

    The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

  5. Evolution of a Membrane Protein Regulon in Saccharomyces

    PubMed Central

    Martin, Hilary C.; Roop, Jeremy I.; Schraiber, Joshua G.; Hsu, Tiffany Y.; Brem, Rachel B.

    2012-01-01

    Expression variation is widespread between species. The ability to distinguish regulatory change driven by natural selection from the consequences of neutral drift remains a major challenge in comparative genomics. In this work, we used observations of mRNA expression and promoter sequence to analyze signatures of selection on groups of functionally related genes in Saccharomycete yeasts. In a survey of gene regulons with expression divergence between Saccharomyces cerevisiae and S. paradoxus, we found that most were subject to variation in trans-regulatory factors that provided no evidence against a neutral model. However, we identified one regulon of membrane protein genes controlled by unlinked cis- and trans-acting determinants with coherent effects on gene expression, consistent with a history of directional, nonneutral evolution. For this membrane protein group, S. paradoxus alleles at regulatory loci were associated with elevated expression and altered stress responsiveness relative to other yeasts. In a phylogenetic comparison of promoter sequences of the membrane protein genes between species, the S. paradoxus lineage was distinguished by a short branch length, indicative of strong selective constraint. Likewise, sequence variants within the S. paradoxus population, but not across strains of other yeasts, were skewed toward low frequencies in promoters of genes in the membrane protein regulon, again reflecting strong purifying selection. Our results support a model in which a distinct expression program for the membrane protein genes in S. paradoxus has been preferentially maintained by negative selection as the result of an increased importance to organismal fitness. These findings illustrate the power of integrating expression- and sequence-based tests of natural selection in the study of evolutionary forces that underlie regulatory change. PMID:22319167

  6. Helical Membrane Protein Conformations and their Environment

    PubMed Central

    Cross, Timothy A.; Murray, Dylan T.; Watts, Anthony

    2013-01-01

    Evidence that membrane proteins respond conformationally and functionally to their environment is gaining pace. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other non-lipid environments. Here, we review the current status of the field, with specific reference to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principals for model refinement. PMID:23996195

  7. Thermostabilisation of membrane proteins for structural studies

    PubMed Central

    Magnani, Francesca; Serrano-Vega, Maria J.; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A.; Tate, Christopher G.

    2017-01-01

    The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs, because it is possible to determine multiple structures of the thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies. PMID:27466713

  8. Polyene antibiotic that inhibits membrane transport proteins.

    PubMed

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains.

  9. Solid-state NMR and Membrane Proteins

    PubMed Central

    Opella, Stanley J.

    2015-01-01

    The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers. PMID:25681966

  10. Solid-state NMR and membrane proteins

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2015-04-01

    The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects of solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers.

  11. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  12. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  13. TRIM proteins in therapeutic membrane repair of muscular dystrophy.

    PubMed

    Alloush, Jenna; Weisleder, Noah

    2013-07-01

    Muscular dystrophy represents a major unmet medical need; only palliative treatments exist for this group of debilitating diseases. Because multiple forms of muscular dystrophy arise from compromised sarcolemmal membrane integrity, a therapeutic approach that can target this loss of membrane function could be applicable to a number of these distinct diseases.One promising therapeutic approach involves the process the cell uses to repair injuries to the plasma membrane. Recent discoveries of genes associated with the membrane repair process provide an opportunity to promote this process as a way to treat muscular dystrophy. One such gene is mitsugumin 53 (MG53), a member of the tripartite motif (TRIM) family of proteins (TRIM72), which is an essential component of the membrane repair pathway in muscle. Recent results indicate that MG53/TRIM72 protein can be directly applied as a therapeutic agent to increase membrane repair capacity of many cell types and treat some aspects of the disease in mouse models of muscular dystrophy. There is great potential for the use of recombinant human MG53 in treating muscular dystrophy and other diseases in which compromised membrane integrity contributes to the disease. Other TRIM family proteins may provide additional targets for therapeutic intervention in similar disease states.

  14. Role of the TRAF Binding Site and NF-κB Activation in Epstein-Barr Virus Latent Membrane Protein 1-Induced Cell Gene Expression

    PubMed Central

    Devergne, Odile; McFarland, Ellen Cahir; Mosialos, George; Izumi, Kenneth M.; Ware, Carl F.; Kieff, Elliott

    1998-01-01

    In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-κB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IκBα blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-κB is an important component of LMP1

  15. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  16. Protein profiles of hatchery egg shell membrane

    USDA-ARS?s Scientific Manuscript database

    Background: Eggshells, which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of m...

  17. Detergents in Membrane Protein Purification and Crystallisation.

    PubMed

    Anandan, Anandhi; Vrielink, Alice

    2016-01-01

    Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers. A number of detergents with varying structure and physicochemical properties are commercially available and can be applied for this purpose. Nevertheless, it is important to choose a detergent that is not only able to extract the membrane protein but also provide an optimal environment while retaining the correct structural and physical properties of the protein molecule. Choosing the best detergent for this task can be made possible by understanding the physical and chemical properties of the different detergents and their interaction with the IMPs. In addition, understanding the mechanism of membrane solubilisation and protein extraction along with crystallisation requirements, if crystallographic studies are going to be undertaken, can help in choosing the best detergent for the purpose. This chapter aims to present the fundamental properties of detergents and highlight information relevant to IMP crystallisation. The first section of the chapter reviews the physicochemical properties of detergents and parameters essential for predicting their behaviour in solution. The second section covers the interaction of detergents with the biologic membranes and proteins followed by their role in membrane protein crystallisation. The last section will briefly cover the types of detergent and their properties focusing on custom designed detergents for membrane protein studies.

  18. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  19. Protein profiles of hatchery egg shell membrane.

    PubMed

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  20. Activation of NFAT-Dependent Gene Expression by Nef: Conservation among Divergent Nef Alleles, Dependence on SH3 Binding and Membrane Association, and Cooperation with Protein Kinase C-θ

    PubMed Central

    Manninen, Aki; Huotari, Päivi; Hiipakka, Marita; Renkema, G. Herma; Saksela, Kalle

    2001-01-01

    Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-θ (PKC-θ) with Nef implicated PKC-θ as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation. PMID:11222731

  1. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

    Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem

    2008-09-23

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  2. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

    Wagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-Willem

    2008-01-01

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. PMID:18796603

  3. The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

    PubMed Central

    Famá, M. Carla; Raden, David; Zacchi, Nicolás; Lemos, Darío R.; Robinson, Anne S.; Silberstein, Susana

    2007-01-01

    YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Δerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Δscj1Δjem1) and in the induction of the unfolded protein response (Δire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER. PMID:17157937

  4. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

    Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

    2012-02-01

    Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

  5. Protein quality control at the plasma membrane

    PubMed Central

    Okiyoneda, Tsukasa; Apaja, Pirjo M.; Lukacs, Gergely L.

    2011-01-01

    Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with critical regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined. PMID:21571517

  6. Protein quality control at the plasma membrane.

    PubMed

    Okiyoneda, Tsukasa; Apaja, Pirjo M; Lukacs, Gergely L

    2011-08-01

    Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  7. [Characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2010].

    PubMed

    Ai, Jun-Hong; Xie, Zheng-De; Liu, Chun-Yan; Gao, Li-Wei; Yan, Jing

    2012-10-01

    To analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012. Polymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2. Type A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively. EBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.

  8. Electrophysiological characterization of membrane transport proteins.

    PubMed

    Grewer, Christof; Gameiro, Armanda; Mager, Thomas; Fendler, Klaus

    2013-01-01

    Active transport in biological membranes has been traditionally studied using a variety of biochemical and biophysical techniques, including electrophysiology. This review focuses on aspects of electrophysiological methods that make them particularly suited for the investigation of transporter function. Two major approaches to electrical recording of transporter activity are discussed: (a) artificial planar lipid membranes, such as the black lipid membrane and solid supported membrane, which are useful for studies on bacterial transporters and transporters of intracellular compartments, and (b) patch clamp and voltage clamp techniques, which investigate transporters in native cellular membranes. The analytical power of these methods is highlighted by several examples of mechanistic studies of specific membrane proteins, including cytochrome c oxidase, NhaA Na(+)/H(+) exchanger, ClC-7 H(+)/Cl(-) exchanger, glutamate transporters, and neutral amino acid transporters. These examples reveal the wealth of mechanistic information that can be obtained when electrophysiological methods are used in combination with rapid perturbation approaches.

  9. Quantification of Detergents Complexed with Membrane Proteins

    PubMed Central

    Chaptal, Vincent; Delolme, Frédéric; Kilburg, Arnaud; Magnard, Sandrine; Montigny, Cédric; Picard, Martin; Prier, Charlène; Monticelli, Luca; Bornert, Olivier; Agez, Morgane; Ravaud, Stéphanie; Orelle, Cédric; Wagner, Renaud; Jawhari, Anass; Broutin, Isabelle; Pebay-Peyroula, Eva; Jault, Jean-Michel; Kaback, H. Ronald; le Maire, Marc; Falson, Pierre

    2017-01-01

    Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies. PMID:28176812

  10. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    PubMed Central

    Grossmann, Guido; Malinsky, Jan; Stahlschmidt, Wiebke; Loibl, Martin; Weig-Meckl, Ina; Frommer, Wolf B.; Opekarová, Miroslava; Tanner, Widmar

    2008-01-01

    In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins. PMID:19064668

  11. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-11-23

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

  12. Conformation of Membrane Proteins: Bacteriorhodopsin

    DTIC Science & Technology

    1991-12-09

    2-0-methoxypolyethylene glycol-N-hydroxy succinimyl carbonate. (MeO-PEG-SC). MeO- PEG-SC was coupled with the purple membrane (PM) of Halobacterium ...transient intermediate with an absorbance maximum of 480-510 nm was also found. RESULTS 1. The growth of the Halobacterium halobium was optimized (e.g

  13. Concentrating membrane proteins using asymmetric traps and AC electric fields.

    PubMed

    Cheetham, Matthew R; Bramble, Jonathan P; McMillan, Duncan G G; Krzeminski, Lukasz; Han, Xiaojun; Johnson, Benjamin R G; Bushby, Richard J; Olmsted, Peter D; Jeuken, Lars J C; Marritt, Sophie J; Butt, Julea N; Evans, Stephen D

    2011-05-04

    Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins. © 2011 American Chemical Society

  14. etramps, a New Plasmodium falciparum Gene Family Coding for Developmentally Regulated and Highly Charged Membrane Proteins Located at the Parasite–Host Cell Interface

    PubMed Central

    Spielmann, Tobias; Fergusen, David J. P.; Beck, Hans-Peter

    2003-01-01

    After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole and develops from morphologically and metabolically distinct ring to trophozoite stages. During these developmental phases, major structural changes occur within the erythrocyte, but neither the molecular events governing this development nor the molecular composition of the parasitophorous vacuole membrane (PVM) is well known. Herein, we describe a new family of highly cationic proteins from P. falciparum termed early transcribed membrane proteins (ETRAMPs). Thirteen members were identified sharing a conserved structure, of which six were found only during ring stages as judged from Northern and Western analysis. Other members showed different stage-specific expression patterns. Furthermore, ETRAMPs were associated with the membrane fractions in Western blots, and colocalization and selective permeabilization studies demonstrated that ETRAMPs were located in the PVM. This was confirmed by immunoelectron microscopy where the PVM and tubovesicular extensions of the PVM were labeled. Early expressed ETRAMPs clearly defined separate PVM domains compared with the negatively charged integral PVM protein EXP-1, suggesting functionally different domains in the PVM with an oppositely charged surface coat. We also show that the dynamic change of ETRAMP composition in the PVM coincides with the morphological changes during development. The P. falciparum PVM is an important structure for parasite survival, and its analysis might provide better understanding of the requirements of intracellular parasites. PMID:12686607

  15. Horizontal Gene Transfer and Assortative Recombination within the Acinetobacter baumannii Clinical Population Provide Genetic Diversity at the Single carO Gene, Encoding a Major Outer Membrane Protein Channel ▿ † ‡

    PubMed Central

    Mussi, María Alejandra; Limansky, Adriana S.; Relling, Verónica; Ravasi, Pablo; Arakaki, Adrián; Actis, Luis A.; Viale, Alejandro M.

    2011-01-01

    We described previously the presence in Acinetobacter baumannii of a novel outer membrane (OM) protein, CarO, which functions as an l-ornithine OM channel and whose loss was concomitant with increased carbapenem resistance among clonally related nosocomial isolates of this opportunistic pathogen. Here, we describe the existence of extensive genetic diversity at the carO gene within the A. baumannii clinical population. The systematic analysis of carO sequences from A. baumannii isolates obtained from public hospitals in Argentina revealed the existence of four highly polymorphic carO variants among them. Sequence polymorphism between the different A. baumannii CarO variants was concentrated in three well-defined protein regions that superimposed mostly to predicted surface-exposed loops. Polymorphism among A. baumannii CarO variants was manifested in differential electrophoretic mobilities, antigenic properties, abilities to form stable oligomeric structures, and l-ornithine influx abilities through the A. baumannii OM under in vivo conditions. Incongruence between the phylogenies of the clinical A. baumannii isolates analyzed and those of the carO variants they harbor suggests the existence of assortative (entire-gene) carO recombinational exchange within the A. baumannii population. Exchange of carO variants possessing differential characteristics mediated by horizontal gene transfer may constitute an A. baumannii population strategy to survive radically changing environmental conditions, such as the leap from inanimate sources to human hosts and vice versa, persistence in a compromised host, and/or survival in health care facilities. PMID:21764928

  16. Intrinsically disordered proteins drive membrane curvature

    PubMed Central

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-01-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures. PMID:26204806

  17. Intrinsically disordered proteins drive membrane curvature

    NASA Astrophysics Data System (ADS)

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-07-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  18. Intrinsically disordered proteins drive membrane curvature.

    PubMed

    Busch, David J; Houser, Justin R; Hayden, Carl C; Sherman, Michael B; Lafer, Eileen M; Stachowiak, Jeanne C

    2015-07-24

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  19. Protein transfer to membranes upon shape deformation

    NASA Astrophysics Data System (ADS)

    Sagis, L. M. C.; Bijl, E.; Antono, L.; de Ruijter, N. C. A.; van Valenberg, H.

    2013-05-01

    Red blood cells, milk fat droplets, or liposomes all have interfaces consisting of lipid membranes. These particles show significant shape deformations as a result of flow. Here we show that these shape deformations can induce adsorption of proteins to the membrane. Red blood cell deformability is an important factor in several diseases involving obstructions of the microcirculatory system, and deformation induced protein adsorption will alter the rigidity of their membranes. Deformation induced protein transfer will also affect adsorption of cells onto implant surfaces, and the performance of liposome based controlled release systems. Quantitative models describing this phenomenon in biomaterials do not exist. Using a simple quantitative model, we provide new insight in this phenomenon. We present data that show convincingly that for cells or droplets with diameters upwards of a few micrometers, shape deformations induce adsorption of proteins at their interface even at moderate flow rates.

  20. ARAMEMNON, a Novel Database for Arabidopsis Integral Membrane Proteins1

    PubMed Central

    Schwacke, Rainer; Schneider, Anja; van der Graaff, Eric; Fischer, Karsten; Catoni, Elisabetta; Desimone, Marcelo; Frommer, Wolf B.; Flügge, Ulf-Ingo; Kunze, Reinhard

    2003-01-01

    A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de. PMID:12529511

  1. Protein separation using an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

    Jou, Ining; Melnikov, Dmitriy; Gracheva, Maria

    Separation of small proteins by charge with a solid-state porous membrane requires control over the protein's movement. Semiconductor membrane has this ability due to the electrically tunable electric potential profile inside the nanopore. In this work we investigate the possibility to separate the solution of two similar sized proteins by charge. As an example, we consider two small globular proteins abundant in humans: insulin (negatively charged) and ubiquitin (neutral). We find that the localized electric field inside the pore either attracts or repels the charged protein to or from the pore wall which affects the delay time before a successful translocation of the protein through the nanopore. However, the motion of the uncharged ubiquitin is unaffected. The difference in the delay time (and hence the separation) can be further increased by the application of the electrolyte bias which induces an electroosmotic flow in the pore. NSF DMR and CBET Grant No. 1352218.

  2. Crystallization of Membrane Proteins by Vapor Diffusion

    PubMed Central

    Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

  3. Protein aggregation in a membrane environment.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya

    2011-01-01

    Biological membranes are featured by a remarkable ability to modulate a wide range of physiological and pathological processes. Of these, protein aggregation is currently receiving the greatest attention, as one type of the ordered protein aggregates, amyloid fibrils, proved to be involved in molecular etiology of a number of fatal diseases. It has been hypothesized that nucleation of amyloid fibrils and toxic action of their precursors is mediated by lipid-protein interactions. Lipid bilayer provides a variety of environments in which aggregated state of polypeptide chain appears to be more thermodynamically favorable than its monomeric form. The major factors responsible for the enhanced self-association propensity of membrane-bound proteins include (i) structural transition of polypeptide chain into aggregation-prone conformation; (ii) protein crowding in a lipid phase; (iii) particular aggregation-favoring orientation and bilayer embedment of the protein molecules. All these factors are considered in the present review with an emphasis being put on the role of electrostatic, hydrophobic, and hydrogen-bonding phenomena in initiating and modulating the protein aggregation on a membrane template. Likewise, we survey the advanced experimental techniques employed for detection and structural characterization of the aggregated species in membrane systems.

  4. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

    Fluorescence spectroscopy is one of the most powerful tools for characterization of a multitude of biological processes. Of these, the phenomenon of protein oligomerization attracts especial interest due to its crucial role in the formation of fibrillar protein aggregates (amyloid fibrils) involved in ethiology of so-called protein misfolding diseases. It is becoming increasingly substantiated that protein fibrillization in vivo can be initiated and modulated at membrane-water interface. All steps of membrane-assisted fibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition of polypeptide chain into a highly aggregation-prone partially folded conformation, assembly of oligomeric nucleus from membrane-bound monomeric species and fiber elongation can be monitored with a mighty family of fluorescence-based techniques. Furthermore, the mechanisms behind cytotoxicity of prefibrillar protein oligomers are highly amenable to fluorescence analysis. The applications of fluorescence spectroscopy to monitoring protein oligomerization in a membrane environment are exemplified and some problems encountered in such kinds of studies are highlighted.

  5. Breaking the barriers in membrane protein crystallography.

    PubMed

    Kang, Hae Joo; Lee, Chiara; Drew, David

    2013-03-01

    As we appreciate the importance of stabilising membrane proteins, the barriers towards their structure determination are being broken down. This change in mindset comes hand-in-hand with more effort placed on developing methods focused at screening for membrane proteins which are naturally stable in detergent solution or improving those that are not so. In practice, however, it is not easy to decide the best strategy to monitor and improve detergent stability, requiring a decision-making process that can be even more difficult for those new to the field. In this review we outline the importance of membrane protein stability with discussions of the stabilisation strategies applied in context with the use of crystallisation scaffolds and the different types of crystallisation methods themselves. Where possible we also highlight areas that we think could push this field forward with emerging technologies, such as X-ray free electron lasers (X-feL), which could have a big impact on the membrane protein structural biology community. We hope this review will serve as a useful guide for those striving to solve structures of both pro- and eukaryotic membrane proteins.

  6. Plasma membrane H(+) -ATPase gene expression, protein level and activity in growing and non-growing regions of barley (Hordeum vulgare) leaves.

    PubMed

    Visnovitz, Tamás; Solti, Adám; Csikós, György; Fricke, Wieland

    2012-04-01

    Plasma membrane proton ATPase (PM-H⁺-ATPase) is the key means through which plant cells energize nutrient uptake and acidify the apoplast. Both of these processes aid cell elongation; yet, it is not known how such a suspected role of the PM-H⁺-ATPase in growth is reflected through changes in its transcript level and activity in grass leaves. In the present study on leaf three of barley, the elongation zone and the emerged blade, which contained fully expanded cells were analyzed. Plasma membranes were isolated and used to assay the activity (ATPase assay) and abundance (western blotting) of PM-H⁺-ATPase protein. Expression of mRNA was quantified using real-time polymerase chain reaction (qPCR). PM-H⁺-ATPase transcript and protein level and activity differed little between growing and non-growing leaf regions when values were related to unit extracted total RNA and cell number, respectively. However, when values were related to unit surface area of plasma membrane, they were more than twice as high in growing compared with non-growing leaf tissue. It is concluded that this higher surface density of PM-H⁺-ATPase activity in growing barley leaf tissue aids apoplast acidification and cell expansion. Copyright © Physiologia Plantarum 2012.

  7. Protein splicing: selfish genes invade cellular proteins.

    PubMed

    Neff, N F

    1993-12-01

    Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

  8. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  9. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  10. Transmembrane protein sorting driven by membrane curvature.

    PubMed

    Strahl, H; Ronneau, S; González, B Solana; Klutsch, D; Schaffner-Barbero, C; Hamoen, L W

    2015-11-02

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  11. Model-building codes for membrane proteins.

    SciTech Connect

    Shirley, David Noyes; Hunt, Thomas W.; Brown, W. Michael; Schoeniger, Joseph S.; Slepoy, Alexander; Sale, Kenneth L.; Young, Malin M.; Faulon, Jean-Loup Michel; Gray, Genetha Anne

    2005-01-01

    We have developed a novel approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only a sparse set of distance constraints, such as those derived from MS3-D, dipolar-EPR and FRET experiments. Algorithms have been written for searching the conformational space of membrane protein folds matching the set of distance constraints, which provides initial structures for local conformational searches. Local conformation search is achieved by optimizing these candidates against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. This results in refined helical bundles to which the interhelical loops and amino acid side-chains are added. Using a set of only 27 distance constraints extracted from the literature, our methods successfully recover the structure of dark-adapted rhodopsin to within 3.2 {angstrom} of the crystal structure.

  12. An N-terminal Domain of Adenovirus Protein VI Fragments Membranes By Inducing Positive Membrane Curvature

    PubMed Central

    Maier, Oana; Galan, Debra L.; Wodrich, Harald; Wiethoff, Christopher M.

    2010-01-01

    Adenovirus (Ad) membrane penetration during cell entry is poorly understood. Here we show that antibodies which neutralize the membrane lytic activity of the Ad capsid protein VI interfere with Ad endosomal membrane penetration. In vitro studies using a peptide corresponding to an N-terminal amphipathic α-helix of protein VI (VI-Φ), as well as other truncated forms of protein VI suggest that VI-Φ is largely responsible for protein VI binding to and lysing of membranes. Additional studies suggest that VI-Φ lies nearly parallel to the membrane surface. Protein VI fragments membranes and induces highly curved structures. Further studies suggest that Protein VI induces positive membrane curvature. These data support a model in which protein VI binds membranes, inducing positive curvature strain which ultimately leads to membrane fragmentation. These results agree with previous observations of Ad membrane permeabilization during cell entry and provide an initial mechanistic description of a nonenveloped virus membrane lytic protein. PMID:20409568

  13. Predicting membrane protein types with bragging learner.

    PubMed

    Niu, Bing; Jin, Yu-Huan; Feng, Kai-Yan; Liu, Liang; Lu, Wen-Cong; Cai, Yu-Dong; Li, Guo-Zheng

    2008-01-01

    The membrane protein type is an important feature in characterizing the overall topological folding type of a protein or its domains therein. Many investigators have put their efforts to the prediction of membrane protein type. Here, we propose a new approach, the bootstrap aggregating method or bragging learner, to address this problem based on the protein amino acid composition. As a demonstration, the benchmark dataset constructed by K.C. Chou and D.W. Elrod was used to test the new method. The overall success rate thus obtained by jackknife cross-validation was over 84%, indicating that the bragging learner as presented in this paper holds a quite high potential in predicting the attributes of proteins, or at least can play a complementary role to many existing algorithms in this area. It is anticipated that the prediction quality can be further enhanced if the pseudo amino acid composition can be effectively incorporated into the current predictor. An online membrane protein type prediction web server developed in our lab is available at http://chemdata.shu.edu.cn/protein/protein.jsp.

  14. The maize brittle 1 gene encodes amyloplast membrane polypeptides.

    PubMed

    Sullivan, T D; Kaneko, Y

    1995-01-01

    A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies. Following affinity purification, the antibodies recognized a set of 38- to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm. Bt1 proteins were not detected with the preimmune antibodies. A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP. At the same time, the ratio of slow- to fast-migrating forms of the protein decreased. During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes. They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery. These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins.

  15. Proteomics characterization of abundant Golgi membrane proteins.

    PubMed

    Bell, A W; Ward, M A; Blackstock, W P; Freeman, H N; Choudhary, J S; Lewis, A P; Chotai, D; Fazel, A; Gushue, J N; Paiement, J; Palcy, S; Chevet, E; Lafrenière-Roula, M; Solari, R; Thomas, D Y; Rowley, A; Bergeron, J J

    2001-02-16

    A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

  16. Identification of Protective Epitopes by Sequencing of the Major Outer Membrane Protein Gene of a Variant Strain of Chlamydia psittaci Serotype 1 (Chlamydophila abortus)

    PubMed Central

    Vretou, Evangelia; Psarrou, Evgenia; Kaisar, Maria; Vlisidou, Isabella; Salti-Montesanto, Viviane; Longbottom, David

    2001-01-01

    Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the family Chlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs. PMID:11119563

  17. Major intrinsic proteins in biomimetic membranes.

    PubMed

    Nielsen, Claus Hélix

    2010-01-01

    Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport

  18. Protein permeation through an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

    Jou, Ining A.; Melnikov, Dmitriy V.; Gracheva, Maria E.

    2016-05-01

    Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein’s dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane-electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions.

  19. Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

    PubMed Central

    Jun, So Hyun; Lee, Jung Hwa; Kim, Bo Ra; Kim, Seung Il; Park, Tae In

    2013-01-01

    Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host. PMID:23977136

  20. Directional interactions and cooperativity between mechanosensitive membrane proteins

    NASA Astrophysics Data System (ADS)

    Haselwandter, Christoph A.; Phillips, Rob

    2013-03-01

    While modern structural biology has provided us with a rich and diverse picture of membrane proteins, the biological function of membrane proteins is often influenced by the mechanical properties of the surrounding lipid bilayer. Here we explore the relation between the shape of membrane proteins and the cooperative function of membrane proteins induced by membrane-mediated elastic interactions. For the experimental model system of mechanosensitive ion channels we find that the sign and strength of elastic interactions depend on the protein shape, yielding distinct cooperative gating curves for distinct protein orientations. Our approach predicts how directional elastic interactions affect the molecular structure, organization, and biological function of proteins in crowded membranes.

  1. Self diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The

  2. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    PubMed

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  3. Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

    PubMed Central

    Cordella, Nicholas; Lampo, Thomas J.; Mehraeen, Shafigh; Spakowitz, Andrew J.

    2014-01-01

    We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures. PMID:24703309

  4. Engineering Lipid Bilayer Membranes for Protein Studies

    PubMed Central

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  5. MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

    PubMed Central

    Stansfeld, Phillip J.; Goose, Joseph E.; Caffrey, Martin; Carpenter, Elisabeth P.; Parker, Joanne L.; Newstead, Simon; Sansom, Mark S.P.

    2015-01-01

    Summary There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein. PMID:26073602

  6. Subdiffusion of proteins and oligomers on membranes

    NASA Astrophysics Data System (ADS)

    Lepzelter, David; Zaman, Muhammad

    2012-11-01

    Diffusion of proteins on lipid membranes plays a central role in cell signaling processes. From a mathematical perspective, most membrane diffusion processes are explained by the Saffman-Delbrück theory. However, recent studies have suggested a major limitation in the theoretical framework, the lack of complexity in the modeled lipid membrane. Lipid domains (sometimes termed membrane rafts) are known to slow protein diffusion, but there have been no quantitative theoretical examinations of how much diffusion is slowed in a general case. We provide an overall theoretical framework for confined-domain ("corralled") diffusion. Further, there have been multiple apparent contradictions of the basic conclusions of Saffman and Delbrück, each involving cases in which a single protein or an oligomer has multiple transmembrane regions passing through a lipid phase barrier. We present a set of corrections to the Saffman-Delbrück theory to account for these experimental observations. Our corrections are able to provide a quantitative explanation of numerous cellular signaling processes that have been considered beyond the scope of the Saffman-Delbrück theory, and may be extendable to other forms of subdiffusion.

  7. Helix-packing motifs in membrane proteins.

    PubMed

    Walters, R F S; DeGrado, W F

    2006-09-12

    The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd membrane proteins.

  8. Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Ramamoorthy, R; Povinelli, L; Philipp M, T

    1996-01-01

    An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete. PMID:8606088

  9. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    PubMed Central

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  10. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    PubMed

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  11. When physics takes over: BAR proteins and membrane curvature

    PubMed Central

    Simunovic, Mijo; Voth, Gregory A.; Callan-Jones, Andrew; Bassereau, Patricia

    2016-01-01

    Cell membranes become highly curved during membrane trafficking, cytokinesis, infection, immune response or cell motion. Bin/amphiphysin/Rvs (BAR) domain proteins with their intrinsically curved and anisotropic shape are involved in many of these processes, but with a large spectrum of modes of action. In vitro experiments and multiscale computer simulations have contributed in identifying a minimal set of physical parameters, namely protein density on the membrane, membrane tension, and membrane shape, that control how bound BAR domain proteins behave on the membrane. In this review, we summarize the multifaceted coupling of BAR proteins to membrane mechanics and propose a simple phase diagram that recapitulates the effects of these parameters. PMID:26519988

  12. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  13. Heterologous expression of membrane proteins: choosing the appropriate host.

    PubMed

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  14. Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

    PubMed

    Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

    2017-02-06

    An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.

  15. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2009-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

  16. Suppressing membrane height fluctuations leads to a membrane-mediated interaction among proteins.

    PubMed

    Sapp, Kayla; Maibaum, Lutz

    2016-11-01

    Membrane-induced interactions can play a significant role in the spatial distribution of membrane-bound proteins. We develop a model that combines a continuum description of lipid bilayers with a discrete particle model of proteins to probe the emerging structure of the combined membrane-protein system. Our model takes into account the membrane's elastic behavior, the steric repulsion between proteins, and the quenching of membrane shape fluctuations due to the presence of the proteins. We employ coupled Langevin equations to describe the dynamics of the system. We show that coupling to the membrane induces an attractive interaction among proteins, which may contribute to the clustering of proteins in biological membranes. We investigate the lateral protein diffusion and find that it is reduced due to transient fluctuations in membrane shape.

  17. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  18. Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins.

    PubMed

    Ferro, M; Seigneurin-Berny, D; Rolland, N; Chapel, A; Salvi, D; Garin, J; Joyard, J

    2000-10-01

    As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid-interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

  19. A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.

    PubMed

    Hu, Jian; Qin, Huajun; Gao, Fei Philip; Cross, Timothy A

    2011-11-01

    Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.

  20. Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane.

    PubMed

    Nothwehr, S F; Conibear, E; Stevens, T H

    1995-04-01

    The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4-ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.

  1. Plasmid-determined resistance to serum bactericidal activity: a major outer membrane protein, the traT gene product, is responsible for plasmid-specified serum resistance in Escherichia coli.

    PubMed Central

    Moll, A; Manning, P A; Timmis, K N

    1980-01-01

    Resistance to the bactericidal activity of serum appears to be an important virulence property of invasive bacteria. The conjugative multiple-antibiotic-resistance plasmid R6-5 was found to confer upon Escherichia coli host bacteria increased resistance against rabbit serum. Gene-cloning techniques were used to localize the serum resistance determinant of R6-5 to a segment of the plasmid that encodes conjugal transfer functions, and a pACYC184 hybrid plasmid, designated pKT107, that contains this segment was constructed. The generation and analysis of deletion and insertion mutant derivatives of the pKT107 plasmid that no longer specify serum resistance permitted precise localization of the serum-resistance cistron on the R6-5 map and demonstrated that this locus is coincident with that of traT, one of the two surface exclusion genes of R6-5. Examination of the proteins synthesized in E. coli minicells of pKT107 and its serum-sensitive mutant derivative plasmids confirmed that the serum-resistance gene product of R6-5 is the traT protein and showed that this protein is a major structural component (about 21,000 copies per cell) of the bacterial outer membrane. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:6995306

  2. Stochastic single-molecule dynamics of synaptic membrane protein domains

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  3. Xanthophylls as modulators of membrane protein function.

    PubMed

    Ruban, Alexander V; Johnson, Matthew P

    2010-12-01

    This review discusses the structural aspect of the role of photosynthetic antenna xanthophylls. It argues that xanthophyll hydrophobicity/polarity could explain the reason for xanthophyll variety and help to understand their recently emerging function--control of membrane organization and the work of membrane proteins. The structure of a xanthophyll molecule is discussed in relation to other amphiphilic compounds like lipids, detergents, etc. Xanthophyll composition of membrane proteins, the role of their variety in protein function are discussed using as an example for the major light harvesting antenna complex of photosystem II, LHCII, from higher plants. A new empirical parameter, hydrophobicity parameter (H-parameter), has been introduced as an effective measure of the hydrophobicity of the xanthophyll complement of LHCII from different xanthophyll biosynthesis mutants of Arabidopsis. Photosystem II quantum efficiency was found to correlate well with the H-parameter of LHCII xanthophylls. PSII down-regulation by non-photochemical chlorophyll fluorescence quenching, NPQ, had optimum corresponding to the wild-type xanthophyll composition, where lutein occupies intrinsic sites, L1 and L2. Xanthophyll polarity/hydrophobicity alteration by the activity of the xanthophyll cycle explains the allosteric character of NPQ regulation, memory of illumination history and the hysteretic nature of the relationship between the triggering factor, ΔpH, and the energy dissipation process. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity.

    PubMed

    Chen, Yih-Yuan; Wu, Chieh-Hao; Lin, Juey-Wen; Weng, Shu-Fen; Tseng, Yi-Hsiung

    2010-09-01

    Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell-cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-beta-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-beta-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

  5. Identification, characterization, and precise mapping of a human gene encoding a novel membrane-spanning protein from the 22q11 region deleted in velo-cardio-facial syndrome.

    PubMed

    Sirotkin, H; Morrow, B; Saint-Jore, B; Puech, A; Das Gupta, R; Patanjali, S R; Skoultchi, A; Weissman, S M; Kucherlapati, R

    1997-06-01

    Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes including cleft palate, conotruncal heart defects, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Using a cDNA selection protocol, we have identified a new gene, TMVCF (transmembrane protein deleted in VCFS), which maps to the deleted interval. The genomic locus is positioned between polymorphic markers D22S944 and D22S941. TMVCF encodes a small protein of 219 amino acids that is predicted to contain two membrane-spanning domains. TMVCF is expressed abundantly in human adult lung, heart, and skeletal muscle, and transcripts can be detected at least as early as Day 9 of mouse development.

  6. J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

    PubMed Central

    D'Silva, Patrick D.; Schilke, Brenda; Walter, William; Andrew, Amy; Craig, Elizabeth A.

    2003-01-01

    The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import. PMID:14605210

  7. J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix.

    PubMed

    D'Silva, Patrick D; Schilke, Brenda; Walter, William; Andrew, Amy; Craig, Elizabeth A

    2003-11-25

    The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.

  8. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

    A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

  9. Functional definition of outer membrane proteins involved in preprotein import into mitochondria.

    PubMed

    Lister, Ryan; Carrie, Chris; Duncan, Owen; Ho, Lois H M; Howell, Katharine A; Murcha, Monika W; Whelan, James

    2007-11-01

    The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

  10. Membrane tension controls the assembly of curvature-generating proteins

    NASA Astrophysics Data System (ADS)

    Simunovic, Mijo; Voth, Gregory A.

    2015-05-01

    Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell.

  11. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions. © 2015 Elsevier Inc. All rights reserved.

  12. Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation.

    PubMed

    Bae, Hyoung Eun; Gotfryd, Kamil; Thomas, Jennifer; Hussain, Hazrat; Ehsan, Muhammad; Go, Juyeon; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

    2015-07-06

    Detergents are an absolute requirement for studying the structure of membrane proteins. However, many conventional detergents fail to stabilise denaturation-sensitive membrane proteins, such as eukaryotic proteins and membrane protein complexes. New amphipathic agents with enhanced efficacy in stabilising membrane proteins will be helpful in overcoming the barriers to studying membrane protein structures. We have prepared a number of deoxycholate-based amphiphiles with carbohydrate head groups, designated deoxycholate-based glycosides (DCGs). These DCGs are the hydrophilic variants of previously reported deoxycholate-based N-oxides (DCAOs). Membrane proteins in these agents, particularly the branched diglucoside-bearing amphiphiles DCG-1 and DCG-2, displayed favourable behaviour compared to previously reported parent compounds (DCAOs) and conventional detergents (LDAO and DDM). Given their excellent properties, these agents should have significant potential for membrane protein studies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Mass spectrometry of membrane proteins: a focus on aquaporins.

    PubMed

    Schey, Kevin L; Grey, Angus C; Nicklay, Joshua J

    2013-06-04

    Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein-protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein-protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins.

  14. Structure Determination of Membrane Proteins in Five Easy Pieces

    PubMed Central

    Marassi, Francesca M.; Das, Bibhuti B.; Lu, George J.; Nothnagel, Henry J.; Park, Sang Ho; Son, Woo Sung; Tian, Ye; Opella, Stanley J.

    2011-01-01

    A general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions is described. Membrane proteins are high priority targets for structure determination, and are challenging for the existing experimental methods. Because membrane proteins reside in a liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, construction and modification of NMR instrumentation, the development and execution of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins which undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. The spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies. PMID:21964394

  15. Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

    PubMed

    Warimwe, George M; Abdi, Abdirahman I; Muthui, Michelle; Fegan, Gregory; Musyoki, Jennifer N; Marsh, Kevin; Bull, Peter C

    2016-05-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.

  16. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    PubMed Central

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  17. Reconstitution of the membrane protein OmpF into biomimetic block copolymer-phospholipid hybrid membranes.

    PubMed

    Bieligmeyer, Matthias; Artukovic, Franjo; Nussberger, Stephan; Hirth, Thomas; Schiestel, Thomas; Müller, Michaela

    2016-01-01

    Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness.

  18. Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

    PubMed Central

    Zilly, Felipe E; Halemani, Nagaraj D; Walrafen, David; Spitta, Luis; Schreiber, Arne; Jahn, Reinhard; Lang, Thorsten

    2011-01-01

    Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid–lipid interactions, lipid–protein interactions and protein–protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca2+ in membrane protein organization. We find that Ca2+ at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca2+ influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca2+ strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes. PMID:21364530

  19. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    PubMed

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  20. Abnormal membrane protein methylation and merocyanine 540 fluorescence in sickle erythrocyte membranes.

    PubMed

    Manna, C; Hermanowicz, N; Ro, J Y; Neilan, B; Glushko, V; Kim, S

    1984-06-01

    Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.

  1. Membrane curvature and its generation by BAR proteins

    PubMed Central

    Mim, Carsten; Unger, Vinzenz M

    2012-01-01

    Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

  2. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains.

    PubMed

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J; Eggeling, Christian; Hell, Stefan W; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-30

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  3. Ankyrin protein networks in membrane formation and stabilization

    PubMed Central

    Cunha, Shane R; Mohler, Peter J

    2009-01-01

    In eukaryotic cells, ankyrins serve as adaptor proteins that link membrane proteins to the underlying cytoskeleton. These adaptor proteins form protein complexes consisting of integral membrane proteins, signalling molecules and cytoskeletal components. With their modular architecture and ability to interact with many proteins, ankyrins organize and stabilize these protein networks, thereby establishing the infrastructure of membrane domains with specialized functions. To this end, ankyrin collaborates with a number of proteins including cytoskeletal proteins, cell adhesion molecules and large structural proteins. This review addresses the targeting and stabilization of protein networks related to ankyrin interactions with the cytoskeletal protein β-spectrin, L1-cell adhesion molecules and the large myofibrillar protein obscurin. The significance of these interactions for differential targeting of cardiac proteins and neuronal membrane formation is also presented. Finally, this review concludes with a discussion about ankyrin dysfunction in human diseases such as haemolytic anaemia, cardiac arrhythmia and neurological disorders. PMID:19840192

  4. Purification of basolateral integral membrane proteins by cationic colloidal silica-based apical membrane subtraction.

    PubMed

    Goode, Robert J A; Simpson, Richard J

    2009-01-01

    Epithelial cell polarity mediates many essential biological functions and perturbation of the apical/basolateral divide is a hallmark of epithelial to mesenchymal transition in carcinoma. Therefore, correct targeting of proteins to the apical and basolateral surfaces is essential to proper epithelial cell function. However, proteomic characterisation of apical/basolateral sorting has been largely ignored, due to ineffectual separation techniques and contamination of plasma-membrane preparations with housekeeping proteins. Here we describe a method that strips the apical membrane from the adherent cells and releases the intracellular contents, thereby leaving the basolateral membrane available for stringent washes and collection. Analysis of the basolateral membrane of an adherent colon adenocarcinoma cell line resulted in 66% of identified proteins being integral membrane proteins, which possessed either a transmembrane domain or lipid modification, including 35 CD antigens. Based on the abundance of peptides from basolateral marker proteins, this method efficiently captures basolateral integral membrane proteins, with minimal contamination from other membranes and basic proteins.

  5. The PURE system for the cell-free synthesis of membrane proteins.

    PubMed

    Kuruma, Yutetsu; Ueda, Takuya

    2015-09-01

    Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

  6. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    PubMed

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  7. Gene Knockout Using Transcription Activator-like Effector Nucleases (TALENs) Reveals That Human NDUFA9 Protein Is Essential for Stabilizing the Junction between Membrane and Matrix Arms of Complex I*

    PubMed Central

    Stroud, David A.; Formosa, Luke E.; Wijeyeratne, Xiaonan W.; Nguyen, Thanh N.; Ryan, Michael T.

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity. PMID:23223238

  8. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

    Membrane curvature has developed into a forefront of membrane biophysics. Numerous proteins involved in membrane curvature sensing and membrane curvature generation have recently been discovered, including proteins containing the crescent-shaped BAR domain as membrane binding and shaping module. Accordingly, the structure determination of these proteins and their multimeric complexes is increasingly well-understood. Substantially less understood, however, are thermodynamic and kinetic aspects and the detailed mechanisms of how these proteins interact with membranes in a curvature-dependent manner. New experimental approaches need to be combined with established techniques to be able to fill in these missing details. Here we use model membrane systems in combination with a variety of biophysical techniques to characterize mechanistic aspects of BAR domain protein function. This includes a characterization of membrane curvature sensing and membrane generation. We also establish kinetic and thermodynamic aspects of BAR protein dimerization in solution, and investigate kinetic aspects of membrane binding. We present two new approaches to investigate membrane shape instabilities and demonstrate that membrane shape instabilities can be controlled by protein binding and lateral membrane tension. This work is supported through NIH grant GM-097552 and NSF grant CBET-1053857.

  9. Identification of yeast genes involved in k homeostasis: loss of membrane traffic genes affects k uptake.

    PubMed

    Fell, Gillian L; Munson, Amanda M; Croston, Merriah A; Rosenwald, Anne G

    2011-06-01

    Using the homozygous diploid Saccharomyces deletion collection, we searched for strains with defects in K(+) homeostasis. We identified 156 (of 4653 total) strains unable to grow in the presence of hygromycin B, a phenotype previously shown to be indicative of ion defects. The most abundant group was that with deletions of genes known to encode membrane traffic regulators. Nearly 80% of these membrane traffic defective strains showed defects in uptake of the K(+) homolog, (86)Rb(+). Since Trk1, a plasma membrane protein localized to lipid microdomains, is the major K(+) influx transporter, we examined the subcellular localization and Triton-X 100 insolubility of Trk1 in 29 of the traffic mutants. However, few of these showed defects in the steady state levels of Trk1, the localization of Trk1 to the plasma membrane, or the localization of Trk1 to lipid microdomains, and most defects were mild compared to wild-type. Three inositol kinase mutants were also identified, and in contrast, loss of these genes negatively affected Trk1 protein levels. In summary, this work reveals a nexus between K(+) homeostasis and membrane traffic, which does not involve traffic of the major influx transporter, Trk1.

  10. Identification of Yeast Genes Involved in K+ Homeostasis: Loss of Membrane Traffic Genes Affects K+ Uptake

    PubMed Central

    Fell, Gillian L.; Munson, Amanda M.; Croston, Merriah A.; Rosenwald, Anne G.

    2011-01-01

    Using the homozygous diploid Saccharomyces deletion collection, we searched for strains with defects in K+ homeostasis. We identified 156 (of 4653 total) strains unable to grow in the presence of hygromycin B, a phenotype previously shown to be indicative of ion defects. The most abundant group was that with deletions of genes known to encode membrane traffic regulators. Nearly 80% of these membrane traffic defective strains showed defects in uptake of the K+ homolog, 86Rb+. Since Trk1, a plasma membrane protein localized to lipid microdomains, is the major K+ influx transporter, we examined the subcellular localization and Triton-X 100 insolubility of Trk1 in 29 of the traffic mutants. However, few of these showed defects in the steady state levels of Trk1, the localization of Trk1 to the plasma membrane, or the localization of Trk1 to lipid microdomains, and most defects were mild compared to wild-type. Three inositol kinase mutants were also identified, and in contrast, loss of these genes negatively affected Trk1 protein levels. In summary, this work reveals a nexus between K+ homeostasis and membrane traffic, which does not involve traffic of the major influx transporter, Trk1. PMID:22384317

  11. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    PubMed

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  12. Large-scale proteomic analysis of membrane proteins

    SciTech Connect

    Ahram, Mamoun; Springer, David L.

    2004-10-01

    Proteomic analysis of membrane proteins is promising in identification of novel candidates as drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solubilization of membrane proteins are frequently encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Unknown proteins are often identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict for the presence of transmembrane domains. Here, we also present these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.

  13. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding.

    PubMed

    De Marothy, Minttu T; Elofsson, Arne

    2015-07-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment. © 2015 The Protein Society.

  14. Staining membrane-bound proteins with coomassie blue r250.

    PubMed

    Stochaj, Wayne R; Berkelman, Tom; Laird, Nancy

    2006-10-01

    INTRODUCTIONCoomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 μg of protein. One drawback of Coomassie Blue staining is that it produces a high background that can make interpretation of results difficult.

  15. Mass Spectrometry of Membrane Proteins: A Focus on Aquaporins

    PubMed Central

    Schey, Kevin L.; Grey, Angus C.; Nicklay, Joshua J.

    2015-01-01

    Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein–protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein–protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins. PMID:23394619

  16. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

  17. Durable vesicles for reconstitution of membrane proteins in biotechnology

    PubMed Central

    Khan, Sanobar; Muench, Stephen P.; Jeuken, Lars J.C.

    2017-01-01

    The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. PMID:28202656

  18. A Prediction Model for Membrane Proteins Using Moments Based Features

    PubMed Central

    Butt, Ahmad Hassan; Khan, Sher Afzal; Jamil, Hamza; Rasool, Nouman; Khan, Yaser Daanial

    2016-01-01

    The most expedient unit of the human body is its cell. Encapsulated within the cell are many infinitesimal entities and molecules which are protected by a cell membrane. The proteins that are associated with this lipid based bilayer cell membrane are known as membrane proteins and are considered to play a significant role. These membrane proteins exhibit their effect in cellular activities inside and outside of the cell. According to the scientists in pharmaceutical organizations, these membrane proteins perform key task in drug interactions. In this study, a technique is presented that is based on various computationally intelligent methods used for the prediction of membrane protein without the experimental use of mass spectrometry. Statistical moments were used to extract features and furthermore a Multilayer Neural Network was trained using backpropagation for the prediction of membrane proteins. Results show that the proposed technique performs better than existing methodologies. PMID:26966690

  19. Charged ultrafiltration membranes increase the selectivity of whey protein separations.

    PubMed

    Bhushan, S; Etzel, M R

    2009-04-01

    Ultrafiltration is widely used to concentrate proteins, but fractionation of one protein from another is much less common. This study examined the use of positively charged membranes to increase the selectivity of ultrafiltration and allow the fractionation of proteins from cheese whey. By adding a positive charge to ultrafiltration membranes, and adjusting the solution pH, it was possible to permeate proteins having little or no charge, such as glycomacropeptide, and retain proteins having a positive charge. Placing a charge on the membrane increased the selectivity by over 600% compared to using an uncharged membrane. The data were fit using the stagnant film model that relates the observed sieving coefficient to membrane parameters such as the flux, mass transfer coefficient, and membrane Peclet number. The model was a useful tool for data analysis and for the scale up of membrane separations for whey protein fractionation.

  20. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    PubMed

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  1. Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

    PubMed Central

    Wen, Yi; Dick, Robert A.

    2016-01-01

    ABSTRACT The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCE Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order

  2. Role of membrane contact sites in protein import into mitochondria.

    PubMed

    Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

    2015-03-01

    Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.

  3. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    PubMed

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (HII) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Dynamic membrane protein topological switching upon changes in phospholipid environment

    PubMed Central

    Vitrac, Heidi; MacLean, David M.; Jayaraman, Vasanthi; Bogdanov, Mikhail; Dowhan, William

    2015-01-01

    A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids. PMID:26512118

  5. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. PMID:25970811

  6. Directional interactions and cooperativity between mechanosensitive membrane proteins

    PubMed Central

    Haselwandter, Christoph A.; Phillips, Rob

    2013-01-01

    While modern structural biology has provided us with a rich and diverse picture of membrane proteins, the biological function of membrane proteins is often influenced by the mechanical properties of the surrounding lipid bilayer. Here we explore the relation between the shape of membrane proteins and the cooperative function of membrane proteins induced by membrane-mediated elastic interactions. For the experimental model system of mechanosensitive ion channels we find that the sign and strength of elastic interactions depend on the protein shape, yielding distinct cooperative gating curves for distinct protein orientations. Our approach predicts how directional elastic interactions affect the molecular structure, organization, and biological function of proteins in crowded membranes. PMID:25309021

  7. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  8. Induction of the lac carrier and an associated membrane protein in Escherichia coli

    SciTech Connect

    Lagarias, D.M.

    1985-01-01

    Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16 kilodalton inner membrane protein in addition to the known products of the lacZ, lacY and lacA genes. Cells carrying the lacY gene on a plasmid over produce this 16 kilodalton polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, (/sup 35/S)methionine labeling of minicells carrying the lacY plasmid shows that the 16 kDa protein is not synthesized from the plasmid DNA. The 16 kDa protein was purified and partially characterized. It is an acidic membrane protein of apparent molecular weight 15,800 whose amino terminal sequence (NH/sub 2/-Met-Arg-Asn-Phe-Asp-Leu-) does not correspond to any nucleotide sequence known in lac operon DNA. Using antibody prepared to the purified 16 kDa protein, a quantitative analysis of conditions under which this protein is made was accomplished, and reveals that the amount of 16 kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligonucleotide probe complementary to the 5' end of 16 kDa protein mRNA shows that its synthesis is regulated at the level of transcription. A description of attempts to clone this gene is given. Possible functional roles for the 16 kDa protein are discussed.

  9. Mapping the yeast host cell response to recombinant membrane protein production: relieving the biological bottlenecks.

    PubMed

    Ashe, Mark P; Bill, Roslyn M

    2011-06-01

    Understanding the structures and functions of membrane proteins is an active area of research within bioscience. Membrane proteins are key players in essential cellular processes such as the uptake of nutrients, the export of waste products, and the way in which cells communicate with their environment. It is therefore not surprising that membrane proteins are targeted by over half of all prescription drugs. Since most membrane proteins are not abundant in their native membranes, it is necessary to produce them in recombinant host cells to enable further structural and functional studies. Unfortunately, achieving the required yields of functional recombinant membrane proteins is still a bottleneck in contemporary bioscience. This has highlighted the need for defined and rational optimization strategies based upon experimental observation rather than relying on trial and error. We have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cells. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yields of recombinant membrane protein: paradoxically, reduced protein synthesis favors higher yields. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

  10. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  11. Fluctuating hydrodynamics of multicomponent membranes with embedded proteins

    SciTech Connect

    Camley, Brian A.; Brown, Frank L. H.

    2014-08-21

    A simulation method for the dynamics of inhomogeneous lipid bilayer membranes is presented. The membrane is treated using stochastic Saffman-Delbrück hydrodynamics, coupled to a phase-field description of lipid composition and discrete membrane proteins. Multiple applications are considered to validate and parameterize the model. The dynamics of membrane composition fluctuations above the critical point and phase separation dynamics below the critical point are studied in some detail, including the effects of adding proteins to the mixture.

  12. Fluctuating hydrodynamics of multicomponent membranes with embedded proteins.

    PubMed

    Camley, Brian A; Brown, Frank L H

    2014-08-21

    A simulation method for the dynamics of inhomogeneous lipid bilayer membranes is presented. The membrane is treated using stochastic Saffman-Delbrück hydrodynamics, coupled to a phase-field description of lipid composition and discrete membrane proteins. Multiple applications are considered to validate and parameterize the model. The dynamics of membrane composition fluctuations above the critical point and phase separation dynamics below the critical point are studied in some detail, including the effects of adding proteins to the mixture.

  13. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  14. Continuum electromechanical modeling of protein-membrane interactions.

    PubMed

    Zhou, Y C; Lu, Benzhuo; Gorfe, Alemayehu A

    2010-10-01

    A continuum electromechanical model is proposed to describe the membrane curvature induced by electrostatic interactions in a solvated protein-membrane system. The model couples the macroscopic strain energy of membrane and the electrostatic solvation energy of the system, and equilibrium membrane deformation is obtained by minimizing the electroelastic energy functional with respect to the dielectric interface. The model is illustrated with the systems with increasing geometry complexity and captures the sensitivity of membrane curvature to the permanent and mobile charge distributions.

  15. cDNA for the human. beta. /sub 2/-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor

    SciTech Connect

    Kobilka, B.K.; Dixon, R.A.F.; Frielle, T.; Dohlman, H.G.; Bolanowski, M.A.; Sigal, I.S.; Yang-Feng, T.L.; Francke, U.; Caron, M.G.; Lefkowitz, R.J.

    1987-01-01

    The authors have isolated and sequenced a cDNA encoding the human ..beta../sub 2/-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster ..beta../sub 2/-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. They have localized the gene for the ..beta../sub 2/-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

  16. A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation

    PubMed Central

    1991-01-01

    The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)- Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like

  17. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

    PubMed

    Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H; McDermott, Ann E

    2015-04-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

  18. Proteomic approaches to identify cold-regulated plasma membrane proteins.

    PubMed

    Takahashi, Daisuke; Nakayama, Takato; Miki, Yushi; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in the plasma membrane proteins have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

  19. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  20. Membrane proteins, lipids and detergents: not just a soap opera.

    PubMed

    Seddon, Annela M; Curnow, Paul; Booth, Paula J

    2004-11-03

    Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant molecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.

  1. High-throughput production of prokaryotic membrane proteins.

    PubMed

    Dobrovetsky, Elena; Lu, Ming Liang; Andorn-Broza, Ronit; Khutoreskaya, Galina; Bray, James E; Savchenko, Alexei; Arrowsmith, Cheryl H; Edwards, Aled M; Koth, Christopher M

    2005-01-01

    Membrane proteins constitute ~30% of prokaryotic and eukaryotic genomes but comprise a small fraction of the entries in protein structural databases. A number of features of membrane proteins render them challenging targets for the structural biologist, among which the most important is the difficulty in obtaining sufficient quantities of purified protein. We are exploring procedures to express and purify large numbers of prokaryotic membrane proteins. A set of 280 membrane proteins from Escherichia coli and Thermotoga maritima, a thermophile, was cloned and tested for expression in Escherichia coli. Under a set of standard conditions, expression could be detected in the membrane fraction for approximately 30% of the cloned targets. About 22 of the highest expressing membrane proteins were purified, typically in just two chromatographic steps. There was a clear correlation between the number of predicted transmembrane domains in a given target and its propensity to express and purify. Accordingly, the vast majority of successfully expressed and purified proteins had six or fewer transmembrane domains. We did not observe any clear advantage to the use of thermophilic targets. Two of the purified membrane proteins formed crystals. By comparison with protein production efforts for soluble proteins, where approximately 70% of cloned targets express and approximately 25% can be readily purified for structural studies [Christendat et al. (2000) Nat. Struct. Biol., 7, 903], our results demonstrate that a similar approach will succeed for membrane proteins, albeit with an expected higher attrition rate.

  2. Phase separation in the isolation and purification of membrane proteins.

    PubMed

    Arnold, Thomas; Linke, Dirk

    2007-10-01

    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.

  3. Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP).

    PubMed

    White, Carl; Nixon, Alex; Bradbury, Neil A

    2015-04-20

    The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

  4. A novel lipoprotein nanoparticle system for membrane proteins

    PubMed Central

    Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A.G.; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

    2016-01-01

    Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state. PMID:26950744

  5. ABCC6 is a Basolateral Plasma Membrane Protein

    PubMed Central

    Pomozi, Viola; Le Saux, Olivier; Brampton, Christopher; Apana, Ailea; Iliás, Attila; Szeri, Flóra; Martin, Ludovic; Monostory, Katalin; Paku, Sándor; Sarkadi, Balázs; Szakács, Gergely; Váradi, András

    2013-01-01

    Rationale ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum (PXE) and general arterial calcification of infancy (GACI). To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective In a recent paper in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane (MAM) and not the plasma membrane. Since the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results We performed immunofluorescent labeling of frozen mouse and human liver sections as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, MAM or the ER. Conclusion Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to the systemic circulation. PMID:23625951

  6. Electrophoretic separation method for membrane pore-forming proteins in multilayer lipid membranes.

    PubMed

    Okamoto, Yukihiro; Tsujimoto, Yusuke; Umakoshi, Hiroshi

    2016-03-01

    In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore-forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high-performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore-forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore-forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore-forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high-performance by utilizing the specific properties of lipid membranes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    PubMed

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  8. Drosophila Golgi membrane protein Ema promotes autophagosomal growth and function.

    PubMed

    Kim, Sungsu; Naylor, Sarah A; DiAntonio, Aaron

    2012-05-01

    Autophagy is a self-degradative process in which cellular material is enclosed within autophagosomes and trafficked to lysosomes for degradation. Autophagosomal biogenesis is well described; however mechanisms controlling the growth and ultimate size of autophagosomes are unclear. Here we demonstrate that the Drosophila membrane protein Ema is required for the growth of autophagosomes. In an ema mutant, autophagosomes form in response to starvation and developmental cues, and these autophagosomes can mature into autolysosomes; however the autophagosomes are very small, and autophagy is impaired. In fat body cells, Ema localizes to the Golgi complex and is recruited to the membrane of autophagosomes in response to starvation. The Drosophila Golgi protein Lva also is recruited to the periphery of autophagosomes in response to starvation, and this recruitment requires ema. Therefore, we propose that Golgi is a membrane source for autophagosomal growth and that Ema facilitates this process. Clec16A, the human ortholog of Ema, is a candidate autoimmune susceptibility locus. Expression of Clec16A can rescue the autophagosome size defect in the ema mutant, suggesting that regulation of autophagosome morphogenesis may be a fundamental function of this gene family.

  9. Surfactant-free purification of membrane proteins with intact native membrane environment.

    PubMed

    Jamshad, Mohammed; Lin, Yu-Pin; Knowles, Timothy J; Parslow, Rosemary A; Harris, Craig; Wheatley, Mark; Poyner, David R; Bill, Roslyn M; Thomas, Owen R T; Overduin, Michael; Dafforn, Tim R

    2011-06-01

    In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

  10. Membrane Protein Production in Lactococcus lactis for Functional Studies.

    PubMed

    Seigneurin-Berny, Daphne; King, Martin S; Sautron, Emiline; Moyet, Lucas; Catty, Patrice; André, François; Rolland, Norbert; Kunji, Edmund R S; Frelet-Barrand, Annie

    2016-01-01

    Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

  11. Chitosan-based membrane chromatography for protein adsorption and separation.

    PubMed

    Liu, Yezhuo; Feng, Zhicheng; Shao, Zhengzhong; Chen, Xin

    2012-08-01

    A chitosan-based membrane chromatography was set up by using natural chitosan/carboxymethylchitosan (CS/CMCS) blend membrane as the matrix. The dynamic adsorption property for protein (lysozyme as model protein) was detailed discussed with the change in pore size of the membrane, the flow rate and the initial concentration of the feed solution, and the layer of membrane in membrane stack. The best dynamic adsorption capacity of lysozyme on the CS/CMCS membrane chromatography was found to be 15.3mg/mL under the optimal flow conditions. Moreover, the CS/CMCS membrane chromatography exhibited good repeatability and reusability with the desorption efficiency of ~90%. As an application, lysozyme and ovalbumin were successfully separated from their binary mixture through the CS/CMCS membrane chromatography. This implies that such a natural chitosan-based membrane chromatography may have great potential on the bioseparation field in the future.

  12. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  13. Challenges in the Development of Functional Assays of Membrane Proteins

    PubMed Central

    Tiefenauer, Louis; Demarche, Sophie

    2012-01-01

    Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  14. An Integrated Framework Advancing Membrane Protein Modeling and Design

    PubMed Central

    Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

    2015-01-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  15. Simple model of membrane proteins including solvent.

    PubMed

    Pagan, D L; Shiryayev, A; Connor, T P; Gunton, J D

    2006-05-14

    We report a numerical simulation for the phase diagram of a simple two-dimensional model, similar to the one proposed by Noro and Frenkel [J. Chem. Phys. 114, 2477 (2001)] for membrane proteins, but one that includes the role of the solvent. We first use Gibbs ensemble Monte Carlo simulations to determine the phase behavior of particles interacting via a square-well potential in two dimensions for various values of the interaction range. A phenomenological model for the solute-solvent interactions is then studied to understand how the fluid-fluid coexistence curve is modified by solute-solvent interactions. It is shown that such a model can yield systems with liquid-liquid phase separation curves that have both upper and lower critical points, as well as closed loop phase diagrams, as is the case with the corresponding three-dimensional model.

  16. Tetra Detector Analysis of Membrane Proteins

    PubMed Central

    Robbins, Rebecca A.; Stroud, Robert M.

    2014-01-01

    Well-characterized membrane protein detergent complexes (PDC) that are pure, homogenous and stable with minimized excess detergent micelles are essential for functional assays and crystallization studies. Procedural steps to measure the mass, size, shape, homogeneity and molecular composition of PDCs and their host detergent micelle using size exclusion chromatography (SEC) with a Viscotek tetra detector array (TDA; absorbance, refractive index, light scattering and viscosity detectors) are presented. The value of starting with a quality PDC sample, the precision and accuracy of the results, and the use of a digital bench top refractometer are emphasized. An alternate and simplified purification and characterization approach using SEC with dual absorbance and refractive index detectors to optimize detergent and lipid concentration while measuring the PDC homogeneity are also described. Applications relative to purification and characterization goals are illustrated as well. PMID:25081744

  17. Detergent-Specific Membrane Protein Crystallization Screens

    NASA Technical Reports Server (NTRS)

    Wiener, Michael

    2007-01-01

    A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

  18. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    PubMed

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  19. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

    PubMed

    Chan, Maia M; Wooden, Jason M; Tsang, Mark; Gilligan, Diana M; Hirenallur-S, Dinesh K; Finney, Greg L; Rynes, Eric; Maccoss, Michael; Ramirez, Julita A; Park, Heon; Iritani, Brian M

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  20. Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

    PubMed Central

    Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

  1. The mesoscopic membrane with proteins (MesM-P) model

    NASA Astrophysics Data System (ADS)

    Davtyan, Aram; Simunovic, Mijo; Voth, Gregory A.

    2017-07-01

    We present the Mesoscopic Membrane with Proteins (MesM-P) model, an extension of a previously developed elastic membrane model for mesoscale simulations of lipid membranes. MesM-P employs a discrete mesoscopic quasi-particle approach to model protein-facilitated shape and topology changes of the lipid membrane on length and time scales inaccessible to all-atom and quasimolecular coarse-grained molecular dynamics simulations. We investigate the ability of MesM-P to model the behavior of large lipid vesicles as a function of bound protein density. We find four distinct mechanisms for protein aggregation on the surface of the membrane, depending on membrane stiffness and protein spontaneous curvature. We also establish a connection between MesM-P and the results of higher resolution coarse-grained molecular dynamics simulations.

  2. The Use of Detergents to Purify Membrane Proteins.

    PubMed

    Orwick-Rydmark, Marcella; Arnold, Thomas; Linke, Dirk

    2016-04-01

    Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included. Copyright © 2016 John Wiley & Sons, Inc.

  3. Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes.

    PubMed Central

    May, S; Harries, D; Ben-Shaul, A

    2000-01-01

    The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic. PMID:11023883

  4. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  5. Discriminating lysosomal membrane protein types using dynamic neural network.

    PubMed

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  6. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    USDA-ARS?s Scientific Manuscript database

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

  7. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    USDA-ARS?s Scientific Manuscript database

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

  8. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  9. Membrane Interacting Regions of Dengue Virus NS2A Protein

    PubMed Central

    2015-01-01

    The Dengue virus (DENV) NS2A protein, essential for viral replication, is a poorly characterized membrane protein. NS2A displays both protein/protein and membrane/protein interactions, yet neither its functions in the viral cycle nor its active regions are known with certainty. To highlight the different membrane-active regions of NS2A, we characterized the effects of peptides derived from a peptide library encompassing this protein’s full length on different membranes by measuring their membrane leakage induction and modulation of lipid phase behavior. Following this initial screening, one region, peptide dens25, had interesting effects on membranes; therefore, we sought to thoroughly characterize this region’s interaction with membranes. This peptide presents an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment. We show that dens25 strongly interacts with membranes that contain a large proportion of lipid molecules with a formal negative charge, and that this effect has a major electrostatic contribution. Considering its membrane modulating capabilities, this region might be involved in membrane rearrangements and thus be important for the viral cycle. PMID:25119664

  10. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    PubMed

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  11. Protein Array-Based Detection of Proteins in Kidney Tissues from Patients with Membranous Nephropathy

    PubMed Central

    Lu, Yang; Fan, Meng

    2017-01-01

    Membranous nephropathy (MN) is an autoimmune inflammatory disease in which proteins related with plenty of biological processes play an important role. However, the role of these proteins in the pathogenesis of MN is still unclear. This study aimed to screen differential proteins in kidney tissue samples from MN patients by using protein arrays and determine the pathways involved in the pathogenesis of MN. This study first tested a quantitative protein array (QAH-INF-3) and two semiquantitative protein arrays (L-493 and L-507) with normal renal tissue and identified L-493 as the most appropriate assay to compare protein levels between MN tissues and normal control tissues. The L-493 array identified 66 differentially expressed proteins (DEPs) that may be associated with MN. The gene oncology (GO) and protein-protein interaction (PPI) analyses revealed several processes potentially involved in MN, including extracellular matrix disassembly and organization, cell adhesion, cell-cell signaling, cellular protein metabolic process, and immune response (P < 0.05). We suggest that these different pathways work together via protein signaling and result in the pathogenesis and progression of MN. PMID:28337458

  12. Construction of a synthetic messenger RNA encoding a membrane protein

    PubMed Central

    1983-01-01

    We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV- infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes. PMID:6341380

  13. Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

    NASA Astrophysics Data System (ADS)

    Mitra, Kakoli; Ubarretxena-Belandia, Iban; Taguchi, Tomohiko; Warren, Graham; Engelman, Donald M.

    2004-03-01

    A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.

  14. Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

    PubMed Central

    Mitra, Kakoli; Ubarretxena-Belandia, Iban; Taguchi, Tomohiko; Warren, Graham; Engelman, Donald M.

    2004-01-01

    A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings. PMID:15016920

  15. Abnormal erythrocyte membrane protein pattern in severe megaloblastic anemia.

    PubMed Central

    Ballas, S K

    1978-01-01

    The erythrocyte membrane protein pattern of patients with megaloblastic anemia was determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In severe megaloblastic anemia, secondary either to folic acid or vitamin B12 deficiency, the erythrocyte membrane protein pattern was grossly abnormal, lacking bands 1, 2 (spectrin), and 3 and having several diffuse, faster migrating bands. After adequate vitamin replacement therapy, the erythrocyte membrane protein pattern returned to normal. In mild megaloblastic anemia, secondary either to folic acid of vitamin B12 deficiency, and in severe iron deficiency anemia, the erythrocyte membrane protein pattern was normal. Erythrocyte membrane protein pattern of normal membranes did not change after mixing with abnormal membranes before polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Protease activity extracted from membranes of megalocytes was not different from normal. These findings indicate that the erythrocyte membrane protein pattern is abnormal in severe megaloblastic anemia and that this abnormality is not secondary to increased activity of the endogenous erythrocyte membrane proteinase. Images PMID:659579

  16. Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

    PubMed

    Amar, A; Rottem, S; Kahane, I; Razin, S

    1976-03-05

    Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

  17. How curvature-generating proteins build scaffolds on membrane nanotubes.

    PubMed

    Simunovic, Mijo; Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T; Lorman, Vladimir; Voth, Gregory A; Bassereau, Patricia

    2016-10-04

    Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube's length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30-40% of a tube's surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.

  18. Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase

    PubMed Central

    Török, Zsolt; Tsvetkova, Nelly M.; Balogh, Gábor; Horváth, Ibolya; Nagy, Enikő; Pénzes, Zoltán; Hargitai, Judit; Bensaude, Olivier; Csermely, Péter; Crowe, John H.; Maresca, Bruno; Vígh, László

    2003-01-01

    The hydroxylamine derivative bimoclomol (BM) has been shown to activate natural cytoprotective homeostatic responses by enhancing the capability of cells to cope with various pathophysiological conditions. It exerts its effect in synergy with low levels of stress to induce the synthesis of members of major stress protein families. We show here that the presence of BM does not influence protein denaturation in the cells. BM and its derivatives selectively interact with acidic lipids and modulate their thermal and dynamic properties. BM acts as a membrane fluidizer at normal temperature, but it is a highly efficient membrane stabilizer, inhibiting the bilayer–nonbilayer phase transitions during severe heat shock. We suggest that BM and the related compounds modify those domains of membrane lipids where the thermally or chemically induced perturbation of lipid phase is sensed and transduced into a cellular signal, leading to enhanced activation of heat shock genes. BM may be a prototype for clinically safe membrane-interacting drug candidates that rebalance the level and composition of heat shock proteins. PMID:12615993

  19. Toward understanding driving forces in membrane protein folding.

    PubMed

    Hong, Heedeok

    2014-12-15

    α-Helical membrane proteins are largely composed of nonpolar residues that are embedded in the lipid bilayer. An enigma in the folding of membrane proteins is how a polypeptide chain can be condensed into the compact folded state in the environment where the hydrophobic effect cannot strongly drive molecular interactions. Probably other forces such as van der Waals packing, hydrogen bonding, and weakly polar interactions, which are regarded less important in the folding of water-soluble proteins, should emerge. However, it is not clearly understood how those individual forces operate and how they are balanced for stabilizing membrane proteins. Studying this problem is not a trivial task mainly because of the methodological challenges in controlling the reversible folding of membrane proteins in the lipid bilayer. Overcoming the hurdles, meaningful progress has been made in the field in the last few decades. This review will focus on recent studies tackling the problem of driving forces in membrane protein folding.

  20. Size-dependent protein segregation at membrane interfaces

    NASA Astrophysics Data System (ADS)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  1. Membrane protein structures without crystals, by single particle electron cryomicroscopy

    PubMed Central

    Vinothkumar, Kutti R

    2015-01-01

    It is an exciting period in membrane protein structural biology with a number of medically important protein structures determined at a rapid pace. However, two major hurdles still remain in the structural biology of membrane proteins. One is the inability to obtain large amounts of protein for crystallization and the other is the failure to get well-diffracting crystals. With single particle electron cryomicroscopy, both these problems can be overcome and high-resolution structures of membrane proteins and other labile protein complexes can be obtained with very little protein and without the need for crystals. In this review, I highlight recent advances in electron microscopy, detectors and software, which have allowed determination of medium to high-resolution structures of membrane proteins and complexes that have been difficult to study by other structural biological techniques. PMID:26435463

  2. Lattice-Boltzmann-based simulations of membrane protein dynamics

    NASA Astrophysics Data System (ADS)

    Sharby, Tyler; Phelps, Ryan; Antonelli, Michael; Kreft Pearce, Jennifer

    2014-11-01

    The cell membrane is a complex structure composed of a phospholipid bilayer and embedded proteins. Recent work has shown that regions of different mobility exist in the membrane due to a variety of factors and that protein motion can be significantly subdiffusive due to the presence of stationary obstacles. We present work that shows that the combination of stationary obstacles and regions of different mobility can lead to aggregation of proteins in certain regions of the cell membrane. The concentration of stationary proteins is below the percolation threshold. The mechanism of this process is hydrodynamically-mediated interactions of diffusing proteins with themselves, as in hydrodynamic memory, and with obstacles.

  3. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4% SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.

  4. Assembly of outer-membrane proteins in bacteria and mitochondria.

    PubMed

    Tommassen, Jan

    2010-09-01

    The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic alpha-helices, integral outer-membrane proteins (OMPs) form beta-barrels. Similar beta-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these beta-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of beta-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial beta-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

  5. Chlamydial polymorphic membrane proteins: regulation, function and potential vaccine candidates

    PubMed Central

    Vasilevsky, Sam; Stojanov, Milos; Greub, Gilbert; Baud, David

    2016-01-01

    Pmps (Polymorphic Membrane Proteins) are a group of membrane bound surface exposed chlamydial proteins that have been characterized as autotransporter adhesins and are important in the initial phase of chlamydial infection. These proteins all contain conserved GGA (I, L, V) and FxxN tetrapeptide motifs in the N-terminal portion of each protein. All chlamydial species express Pmps. Even in the chlamydia-related bacteria Waddlia chondrophila, a Pmp-like adhesin has been identified, demonstrating the importance of Pmps in Chlamydiales biology. Chlamydial species vary in the number of pmp genes and their differentially regulated expression during the infectious cycle or in response to stress. Studies have also demonstrated that Pmps are able to induce innate immune functional responses in infected cells, including production of IL-8, IL-6 and MCP-1, by activating the transcription factor NF-κB. Human serum studies have indicated that although anti-Pmp specific antibodies are produced in response to a chlamydial infection, the response is variable depending on the Pmp protein. In C. trachomatis, PmpB, PmpC, PmpD and PmpI were the proteins eliciting the strongest immune response among adolescents with and without pelvic inflammatory disease (PID). In contrast, PmpA and PmpE elicited the weakest antibody response. Interestingly, there seems to be a gender bias for Pmp recognition with a stronger anti-Pmp reactivity in male patients. Furthermore, anti-PmpA antibodies might contribute to adverse pregnancy outcomes, at least among women with PID. In vitro studies indicated that dendritic cells infected with C. muridarum were able to present PmpG and PmpF on their MHC class II receptors and T cells were able to recognize the MHC class-II bound peptides. In addition, vaccination with PmpEFGH and Major Outer Membrane Protein (MOMP) significantly protected mice against a genital tract C. muridarum infection, suggesting that Pmps may be an important component of a multi

  6. The conserved carboxyl domain of MorC, an inner membrane protein of Aggregatibacter actinomycetemcomitans, is essential for membrane function.

    PubMed

    Smith, K P; Voogt, R D; Ruiz, T; Mintz, K P

    2016-02-01

    Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C-terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C-terminus of the protein is essential for maintaining membrane physiology.

  7. Membrane Targeting Properties of a Herpesvirus Tegument Protein-Retrovirus Gag Chimera

    PubMed Central

    Bowzard, J. Bradford; Visalli, Robert J.; Wilson, Carol B.; Loomis, Joshua S.; Callahan, Eric M.; Courtney, Richard J.; Wills, John W.

    2000-01-01

    The retroviral Gag protein is capable of directing the production and release of virus-like particles in the absence of all other viral components. Budding normally occurs after Gag is transported to the plasma membrane by its membrane-targeting and -binding (M) domain. In the Rous sarcoma virus (RSV) Gag protein, the M domain is contained within the first 86 amino acids. When M is deleted, membrane association and budding fail to occur. Budding is restored when M is replaced with foreign membrane-binding sequences, such as that of the Src oncoprotein. Moreover, the RSV M domain is capable of targeting heterologous proteins to the plasma membrane. Although the solution structure of the RSV M domain has been determined, the mechanism by which M specifically targets Gag to the plasma membrane rather than to one or more of the large number of internal membrane surfaces (e.g., the Golgi apparatus, endoplasmic reticulum, and nuclear, mitochondrial, or lysosomal membranes) is unknown. To further investigate the requirements for targeting proteins to discrete cellular locations, we have replaced the M domain of RSV with the product of the unique long region 11 (UL11) gene of herpes simplex virus type 1. This 96-amino-acid myristylated protein is thought to be involved in virion transport and envelopment at internal membrane sites. When the first 100 amino acids of RSV Gag (including the M domain) were replaced by the entire UL11 sequence, the chimeric protein localized at and budded into the Golgi apparatus rather than being targeted to the plasma membrane. Myristate was found to be required for this specific targeting, as were the first 49 amino acids of UL11, which contain an acidic cluster motif. In addition to shedding new light on UL11, these experiments demonstrate that RSV Gag can be directed to internal cellular membranes and suggest that regions outside of the M domain do not contain a dominant plasma membrane-targeting motif. PMID:10954570

  8. How curvature-generating proteins build scaffolds on membrane nanotubes

    PubMed Central

    Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T.; Lorman, Vladimir; Voth, Gregory A.; Bassereau, Patricia

    2016-01-01

    Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

  9. Disturbed vesicular trafficking of membrane proteins in prion disease

    PubMed Central

    Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

    2013-01-01

    The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases. PMID:24335150

  10. Disturbed vesicular trafficking of membrane proteins in prion disease.

    PubMed

    Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

    2013-01-01

    The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

  11. Probing Single Membrane Proteins by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Scheuring, S.; Sapra, K. Tanuj; Müller, Daniel J.

    In this book chapter, we describe the working principle of the atomic force microscope (AFM), followed by the applications of AFM in high-resolution imaging and single-molecule force spectroscopy of membrane proteins. In the imaging mode, AFM allows observing the assembly of membrane proteins directly in native membranes approaching a resolution of ~0.5 nm with an outstanding signal-to-noise ratio. Conformational deviations of individual membrane proteins can be observed and their functional states directly imaged. Time-lapse AFM can image membrane proteins at work. In conjunction with high- resolution imaging, the use of the AFM as a single-molecule force spectroscope (SMFS) has gained tremendous importance in recent years. This combination allows to locate the inter- and intramolecular interactions of single membrane proteins. SMFS allows characterization of interactions that guide the folding of proteins and describe the parameters that lead to their destabilization, malfunction and misfolding. Moreover, it enables to measure the interactions established by ligand- and inhibitor-binding and in membrane protein assemblies. Because of its practical use in characterizing various parameters of membrane proteins in their native environment, AFM can be aptly described as a `lab on a tip' device.

  12. Separation of the outer membrane and identification of major outer membrane proteins from Porphyromonas gingivalis.

    PubMed

    Murakami, Yukitaka; Imai, Masashi; Nakamura, Hiroshi; Yoshimura, Fuminobu

    2002-04-01

    The outer membrane of Porphyromonas gingivalis, an oral strict anaerobe, was isolated by sucrose density gradient centrifugation. The outer membrane obtained by the differential detergent extraction method, previously reported, showed an essentially similar protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirming that the latter method is suitable for the study of outer membrane proteins in this organism. N-terminal amino acid sequence analysis revealed that major outer membrane proteins in this organism included Arg-gingipain, Lys-gingipain, RagA (a TonB-linked receptor), and putative porins that were homologous to Escherichia coli OmpA.

  13. Hen oviduct signal peptidase is an integral membrane protein.

    PubMed

    Lively, M O; Walsh, K A

    1983-08-10

    Membrane preparations from rough endoplasmic reticulum of hen oviduct resemble those of dog pancreas in their capacity to translocate nascent secretory proteins into membrane vesicles present during cell-free protein synthesis. As with the dog membranes, the precursor form of human placental lactogen is transported into the vesicles and processed to the native secretory form by an associated "signal peptidase." The oviduct microsomal membranes glycosylate nascent ovomucoid and ovalbumin in vitro. Attempts to extract the signal peptidase from these membrane vesicles revealed that it is one of the least easily solubilized proteins. A protocol for enrichment of signal peptidase was developed that took advantage of its tight association with these vesicles. These studies indicate that the enzyme has the characteristics of an integral membrane protein which remains active in membrane vesicles even after extraction with low concentrations of detergent that do not dissolve the lipid bilayer or after disruption of membrane vesicles in ice-cold 0.1 M Na2CO3, pH 11.5 (Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P.B. (1982) J. Cell Biol. 93, 97-102), which releases the majority of membrane-associated proteins. Solubilization requires concentrations of nondenaturing detergents that totally dissolve the lipid bilayer. The detergent-solubilized enzyme retains the activity and the characteristic specificity of the membrane-bound form.

  14. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  15. Pathogen receptor discovery with a microfluidic human membrane protein array.

    PubMed

    Glick, Yair; Ben-Ari, Ya'ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella; Gerber, Doron

    2016-04-19

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

  16. A sliding selectivity scale for lipid binding to membrane proteins

    PubMed Central

    Landreh, Michael; Marty, Michael T.; Gault, Joseph; Robinson, Carol V.

    2017-01-01

    Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins that reside in the membrane have co-evolved with their hydrophobic lipid environment which serves as a solvent for proteins with very diverse requirements. As a result, membrane protein-lipid interactions range from completely non-selective to highly discriminating. Mass spectrometry (MS), in combination with X-ray crystallography and molecular dynamics simulations, enables us to monitor how lipids interact with intact membrane protein complexes and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. In this review, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization. PMID:27155089

  17. Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins.

    PubMed

    Carroll, Joe; Fearnley, Ian M; Walker, John E

    2006-10-31

    The covalent structure of a protein is incompletely defined by its gene sequence, and mass spectrometric analysis of the intact protein is needed to detect the presence of any posttranslational modifications. Because most membrane proteins are purified in detergents that are incompatible with mass spectrometric ionization techniques, this essential measurement has not been made on many hydrophobic proteins, and so proteomic data are incomplete. We have extracted membrane proteins from bovine mitochondria and detergent-purified NADH:ubiquinone oxidoreductase (complex I) with organic solvents, fractionated the mixtures by hydrophilic interaction chromatography, and measured the molecular masses of the intact membrane proteins, including those of six subunits of complex I that are encoded in mitochondrial DNA. These measurements resolve long-standing uncertainties about the interpretation of the mitochondrial genome, and they contribute significantly to the definition of the covalent composition of complex I.

  18. Curvature Forces in Membrane Lipid-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Brown, Michael F.

    2012-02-01

    Membrane protein conformational changes, folding, and stability may all involve elastic deformation of the bilayer. Non-specific properties of the bilayer play a significant role in modulating protein conformational energetics. A flexible-surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. The FSM describes elastic coupling of membrane lipids to integral membrane proteins. Curvature and hydrophobic matching to the lipid bilayer entails a stress field that explains membrane protein stability. Rhodopsin provides an important example, where solid-state NMR and FTIR spectroscopy characterize the energy landscape of the dynamically activated receptor. Time-resolved UV-visible and FTIR spectroscopic studies show how membrane lipids affect the metarhodopsin equilibrium due to non-specific material properties. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on rhodopsin function are all explained by the new biomembrane model. By contrast, the older fluid-mosaic model fails to account for such effects on membrane protein activity. According to the FSM proteins are regulated by membrane lipids whose spontaneous curvature most closely matches the activated state within the lipid membrane.

  19. Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

    PubMed Central

    Snider, Jamie; Kittanakom, Saranya; Curak, Jasna; Stagljar, Igor

    2010-01-01

    The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms. PMID:20125081

  20. Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes

    PubMed Central

    Krumpe, Katrin; Frumkin, Idan; Herzig, Yonatan; Rimon, Nitzan; Özbalci, Cagakan; Brügger, Britta; Rapaport, Doron; Schuldiner, Maya

    2012-01-01

    Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in ∆spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in ∆spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell. PMID:22918956

  1. BPROMPT: A consensus server for membrane protein prediction.

    PubMed

    Taylor, Paul D; Attwood, Teresa K; Flower, Darren R

    2003-07-01

    Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT.

  2. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

  3. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom

    PubMed Central

    Xu, Yanjie; Liu, Suxuan; Xia, Jixiang; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2016-01-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair. PMID:28199211

  4. Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

    PubMed Central

    Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

    2011-01-01

    In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

  5. Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

    PubMed

    Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O; Wieschhaus, Adam J; Hartwig, John; Huang, Sha; Han, Jongyoon; Chishti, Athar H

    2016-07-07

    Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. © 2016 by The American Society of Hematology.

  6. Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia

    PubMed Central

    Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O.; Wieschhaus, Adam J.; Hartwig, John; Huang, Sha; Han, Jongyoon

    2016-01-01

    Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin–actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin’s actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate–dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. PMID:27073223

  7. Glycosomal membrane proteins and lipids from Leishmania mexicana.

    PubMed

    Quiñones, Wilfredo; Cáceres, Ana J; Ruiz, Maria Tibisay; Concepción, Juan Luis

    2015-04-01

    Constituents of the glycosomal membrane from Leishmania mexicana should play a critical role in the coordination of metabolic processes occurring in the cytosol and those compartmentalized within glycosomes. We have made an inventory of glycosomal membrane-associated proteins using approaches specific for enriching both integral and peripheral membrane proteins. Surprisingly, 70% of the proteins were recovered in the hydrophobic fraction of membranes solubilized with Triton X-114, while 20% were present in the soluble fraction obtained upon treatment with Na2CO3. 14 major polypeptides, ranging in molecular weight from 65 to 16 kDa, were found to be associated with the membrane, nine of them behaving as integral membrane proteins. Assessment of their topology in the membrane indicated that the polypeptides of 56, 50, 46 and 32 kDa have no domains exposed to the cytosol. The 50 kDa protein is the most abundant one of the glycosomal membrane, where it is peripherically located at the matrix face. The major phospholipids of glycosomal membranes are phosphatidyl-ethanolamine, phosphatidyl-choline and phosphatidyl-serine, with smaller proportions of sphingomyelin and phosphatidyl-inositol. The sterols found were of 5-dehydroepisterol, ergosta-5,7,24(24(1))-trien-3β-ol, and also their precursors, consistent with the notion that these organelles are involved in de novo biosynthesis of sterols in trypanosomatids. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy.

    PubMed

    Bronder, Anna M; Bieker, Adeline; Elter, Shantha; Etzkorn, Manuel; Häussinger, Dieter; Oesterhelt, Filipp

    2016-11-01

    Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Applications of solid-state NMR to membrane proteins.

    PubMed

    Ladizhansky, Vladimir

    2017-07-12

    Membrane proteins mediate flow of molecules, signals, and energy between cells and intracellular compartments. Understanding membrane protein function requires a detailed understanding of the structural and dynamic properties involved. Lipid bilayers provide a native-like environment for structure-function investigations of membrane proteins. In this review we give a general discourse on the recent progress in the field of solid-state NMR of membrane proteins. Solid-state NMR is a variation of NMR spectroscopy that is applicable to molecular systems with restricted mobility, such as high molecular weight proteins and protein complexes, supramolecular assemblies, or membrane proteins in a phospholipid environment. We highlight recent advances in applications of solid-state NMR to membrane proteins, specifically focusing on the recent developments in the field of Dynamic Nuclear Polarization, proton detection, and solid-state NMR applications in situ (in cell membranes). This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Membrane protein production in Escherichia coli cell-free lysates.

    PubMed

    Henrich, Erik; Hein, Christopher; Dötsch, Volker; Bernhard, Frank

    2015-07-08

    Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

  11. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  12. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  13. Misfolding of amyloidogenic proteins and their interactions with membranes.

    PubMed

    Relini, Annalisa; Marano, Nadia; Gliozzi, Alessandra

    2013-12-27

    In this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. Many of these proteins are implicated in serious illnesses such as Alzheimer's disease and Parkinson's disease. Misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. Oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membrane damage. We focus on the structure of these aggregates and their interactions with model membranes. Study of interactions of amlyoidogenic proteins with model and natural membranes has shown the importance of the lipid bilayer in protein misfolding and aggregation and has led to the development of several models for membrane permeabilization by the resulting amyloid aggregates. We discuss several of these models: formation of structured pores by misfolded amyloidogenic proteins, extraction of lipids, interactions with receptors in biological membranes, and membrane destabilization by amyloid aggregates perhaps analogous to that caused by antimicrobial peptides.

  14. Membrane gene ontology bias in sequencing and microarray obtained by housekeeping-gene analysis.

    PubMed

    Zhang, Yijuan; Akintola, Oluwafemi S; Liu, Ken J A; Sun, Bingyun

    2016-01-10

    Microarray (MA) and high-throughput sequencing are two commonly used detection systems for global gene expression profiling. Although these two systems are frequently used in parallel, the differences in their final results have not been examined thoroughly. Transcriptomic analysis of housekeeping (HK) genes provides a unique opportunity to reliably examine the technical difference between these two systems. We investigated here the structure, genome location, expression quantity, microarray probe coverage, as well as biological functions of differentially identified human HK genes by 9 MA and 6 sequencing studies. These in-depth analyses allowed us to discover, for the first time, a subset of transcripts encoding membrane, cell surface and nuclear proteins that were prone to differential identification by the two platforms. We hope that the discovery can aid the future development of these technologies for comprehensive transcriptomic studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Metaproteomic analysis of biocake proteins to understand membrane fouling in a submerged membrane bioreactor.

    PubMed

    Zhou, Zhongbo; Meng, Fangang; He, Xiang; Chae, So-Ryong; An, Yujia; Jia, Xiaoshan

    2015-01-20

    Metaproteomic analyses, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/TOF mass spectrometer (MS) detection, were used to trace and identify biocake proteins on membranes in a bench-scale submerged membrane bioreactor (MBR). 2D-PAGE images showed that proteins in the biocake (S3) at a low transmembrane pressure (TMP) level (i.e., before the TMP jump) had larger gray intensities in the pH 5.5–7.0 region regardless of the membrane flux, similar to soluble microbial product (SMP) proteins. However, the biocake (S2 and S4) at a high TMP level (i.e., after the TMP jump) had many more proteins in the pH range of 4.0–5.5, similar to extracellular polymeric substance (EPS) proteins. Such similarities between biocake proteins and SMP or EPS proteins can be useful for tracing the sources of proteins resulting in membrane fouling. In total, 183 differentially abundant protein spots were marked in the three biocakes (S2, S3, and S4). However, only 32 protein spots co-occurred in the 2D gels of the three biocakes, indicating that membrane fluxes and TMP evolution levels had significant effects on the abundance of biocake proteins. On the basis of the MS and MS/MS data, 23 of 71 protein spots were successfully identified. Of the 23 proteins, outer membrane proteins (Omp) were a major contributor (60.87%). These Omps were mainly from potential surface colonizers such as Aeromonas, Enterobacter, Pseudomonas, and Thauera. Generally, the metaproteomic analysis is a useful alternative to trace the sources and compositions of biocake proteins on the levels of molecules and bacteria species that can provide new insight into membrane fouling.

  16. Membrane protein insertion: mixing eukaryotic and prokaryotic concepts.

    PubMed

    Schleiff, Enrico; Soll, Jürgen

    2005-11-01

    Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.

  17. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey V.; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F.

    2016-01-01

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins. PMID:26908053

  18. Amyloid Aggregation and Membrane Disruption by Amyloid Proteins

    NASA Astrophysics Data System (ADS)

    Ramamoorthy, Ayyalusamy

    2013-03-01

    Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.

  19. Protein-membrane interactions: blood clotting on nanoscale bilayers.

    PubMed

    Morrissey, J H; Pureza, V; Davis-Harrison, R L; Sligar, S G; Rienstra, C M; Kijac, A Z; Ohkubo, Y Z; Tajkhorshid, E

    2009-07-01

    The clotting cascade requires the assembly of protease-cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein-membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease-cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations allow structural insights into interactions between proteins and membrane surfaces with atomic resolution.

  20. Protein-membrane interactions: Blood clotting on nanoscale bilayers

    PubMed Central

    Morrissey, J.H.; Pureza, V.; Davis-Harrison, R.L.; Sligar, S.G.; Rienstra, C.M.; Kijac, A.Z.; Ohkubo, Y. Z.; Tajkhorshid, E.

    2010-01-01

    Summary The clotting cascade requires the assembly of protease-cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein-membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease-cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations are allowing structural insights into interactions between proteins and membrane surfaces with atomic resolution. PMID:19630793

  1. Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

    PubMed

    Fox, Philip D; Haberkorn, Christopher J; Weigel, Aubrey V; Higgins, Jenny L; Akin, Elizabeth J; Kennedy, Matthew J; Krapf, Diego; Tamkun, Michael M

    2013-09-01

    In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

  2. Quenching of fluorescence in membrane protein by hypocrellin B.

    PubMed

    Yue, J; Pang, S

    1997-04-01

    The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical charactcristics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quenchtr between membrane and water, and the fluorescence quenching constant of protein (K(sv); K(q),). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was observed in detail by using the ESR technique. The signal of HB- was found to arise from an electron transfer from excited trytophan to HB.

  3. Host membrane proteins involved in the replication of tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Miyashita, Shuhei; Katoh, Etsuko; Ishikawa, Masayuki

    2012-12-01

    Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

  4. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  5. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  6. Protein kinase and phosphatase activities of thylakoid membranes

    SciTech Connect

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg/sup 2 +/ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg/sup 2 +/ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs.

  7. Surface-Bound Membrane-Mimetic Assemblies: Electrostatic Attributes of Integral Membrane Proteins

    DTIC Science & Technology

    1988-10-31

    other than bovine rhodopsin, we have used analogous techniques with a second retinal-containing protein, bacteriorhodopsin . This protein is found in the...purple membrane of Halobacterium halobium and serves as a light-driven proton pump to generate a transmembrane proton gradient used by the bacterium...and characterized by its absorption spectra. Figure 3 compares the spectra of bacteriorhodopsin in the natural purple membrane with that from protein

  8. MALDI tissue profiling of integral membrane proteins from ocular tissues.

    PubMed

    Thibault, Danielle B; Gillam, Christopher J; Grey, Angus C; Han, Jun; Schey, Kevin L

    2008-06-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this article, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed age related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods.

  9. Predicting residue and helix contacts in membrane proteins

    NASA Astrophysics Data System (ADS)

    Fuchs, Angelika; Kirschner, Andreas; Frishman, Dmitrij

    Helix-helix contacts are an important feature of alpha-helical membrane proteins as they define their characteristic helix bundle structure. No bioinformatics approaches for the prediction of pairwise residue contacts in membrane proteins have existed until recently. In this chapter we describe novel contact prediction methods based on residue coevolution and machine learning techniques specifically geared towards membrane proteins. While contact prediction accuracies are limited to ~10% using co-evolving residues alone, machine learning methods are able to improve these accuracies significantly to more than 25% by using available membrane protein structures as a training dataset and incorporating membrane protein specific sequence features into the prediction process. Importantly, predicted residue contacts allow for identification of interacting transmembrane helices with high accuracy. As different membrane protein structures can be distinguished by their specific pattern of helix interactions, predicted residue contacts may not only serve as structural constraints in modeling experiments, but also constitute valuable information for structural classification of membrane proteins with unknown structure.

  10. β-Barrel membrane protein assembly by the Bam complex.

    PubMed

    Hagan, Christine L; Silhavy, Thomas J; Kahne, Daniel

    2011-01-01

    β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

  11. Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma.

    PubMed

    Płaszczyca, Anna; Nilsson, Jenny; Magnusson, Linda; Brosjö, Otte; Larsson, Olle; Vult von Steyern, Fredrik; Domanski, Henryk A; Lilljebjörn, Henrik; Fioretos, Thoas; Tayebwa, Johnbosco; Mandahl, Nils; Nord, Karolin H; Mertens, Fredrik

    2014-08-01

    Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.

  12. Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

    PubMed Central

    Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

    2017-01-01

    Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

  13. Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing.

    PubMed Central

    Hincha, Dirk K

    2002-01-01

    Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage. PMID:12171654

  14. Yeast mitochondrial fission proteins induce antagonistic Gaussian membrane curvatures to regulate apoptosis

    NASA Astrophysics Data System (ADS)

    Lee, Michelle; Hwee Lai, Ghee; Schmidt, Nathan; Xian, Wujing; Wong, Gerard C. L.

    2013-03-01

    Mitochondria form a dynamic and interconnected network, which disintegrates during apoptosis to generate numerous smaller mitochondrial fragments. This process is at present not well understood. Yeast mitochondrial fission machinery proteins, Dnm1 and Fis1, are believed to regulate programmed cell death in yeast. Yeast Dnm1 has been previously shown to promote mitochondrial fragmentation and degradation characteristic of apoptotic cells, while yeast Fis1 inhibits cell death by limiting the mitochondrial fission induced by Dnm1 [Fannjiang et al, Genes & Dev. 2004. 18: 2785-2797]. To better understand the mechanisms of these antagonistic fission proteins, we use synchrotron small angle x-ray scattering (SAXS) to investigate their interaction with model cell membranes. The relationship between each protein, Dnm1 and Fis1, and protein-induced changes in membrane curvature and topology is examined. Through the comparison of the membrane rearrangement and phase behavior induced by each protein, we will discuss their respective roles in the regulation of mitochondrial fission.

  15. Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm.

    PubMed Central

    Schneider, D L; Burnside, J; Gorga, F R; Nettleton, C J

    1978-01-01

    Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances. PMID:153136

  16. Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

    PubMed

    Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

    2015-04-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  17. Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

    PubMed Central

    Tiner, Bethany L.; Kirtley, Michelle L.; Erova, Tatiana E.; Popov, Vsevolod L.; Baze, Wallace B.; van Lier, Christina J.; Ponnusamy, Duraisamy; Andersson, Jourdan A.; Motin, Vladimir L.; Chauhan, Sadhana

    2015-01-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  18. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    PubMed

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  19. Genetically Encoded Protein Sensors of Membrane Potential.

    PubMed

    Storace, Douglas; Rad, Masoud Sepehri; Han, Zhou; Jin, Lei; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J; Sung, Uhna

    2015-01-01

    Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. However these dyes suffer from the drawbacks of non-specificity of cell staining and low accessibility of the dye to some cell types. Further progress in imaging activity is expected from the development of genetically encoded fluorescent sensors of membrane potential. Cell type specificity of expression of these fluorescent protein (FP) voltage sensors can be obtained via several different mechanisms. One is cell type specificity of infection by individual virus subtypes. A second mechanism is specificity of promoter expression in individual cell types. A third, depends on the offspring of transgenic animals with cell type specific expression of cre recombinase mated with an animal that has the DNA for the FP voltage sensor in all of its cells but its expression is dependent on the recombinase activity. Challenges remain. First, the response time constants of many of the new FP voltage sensors are slower (2-10 ms) than those of organic dyes. This results in a relatively small fractional fluorescence change, ΔF/F, for action potentials. Second, the largest signal presently available is only ~40% for a 100 mV depolarization and many of the new probes have signals that are substantially smaller. Large signals are especially important when attempting to detect fast events because the shorter measurement interval results in a relatively small number of detected photons and therefore a relatively large shot noise (see Chap. 1). Another kind of challenge has occurred when attempts were made to transition from one species to another or from one cell type to another or from cell culture to in vivo measurements.Several laboratories have recently described a number of novel FP voltage sensors. Here we attempt to critically review the current status of these developments in terms of signal size, time course, and in vivo function.

  20. Chromatin Architecture and Transcription Factor Binding Regulate Expression of Erythrocyte Membrane Protein Genes▿ †

    PubMed Central

    Steiner, Laurie A.; Maksimova, Yelena; Schulz, Vincent; Wong, Clara; Raha, Debasish; Mahajan, Milind C.; Weissman, Sherman M.; Gallagher, Patrick G.

    2009-01-01

    Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia. PMID:19687298

  1. The electrical interplay between proteins and lipids in membranes.

    PubMed

    Richens, Joanna L; Lane, Jordan S; Bramble, Jonathan P; O'Shea, Paul

    2015-09-01

    All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure. The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid-protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid-protein interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  3. Mapping of four mouse genes encoding eye lens-specific structural, gap junction, and integral membrane proteins: Cryba1 (crystallin{beta}A3/A1), Crybb2 (crystallin{beta}B2), Gja8 (MP70), and Lim2 (MP19)

    SciTech Connect

    Kerscher, S.; Boyd, Y.; Lyon, M.F.

    1995-09-20

    Four genes encoding eye lens-specific proteins, potential candidate genes for congenital cataract (CC) mutations, were mapped in the mouse genome using a panel of somatic cell hybrids and DNAs from the EUCIB (European Collaborative Interspecific Backcross). Two of them are lens fiber cell structural proteins: the Cryba1 locus encoding crystallin{beta}A3/A1 maps to chromosome 11, 2.5 {+-} 2.5 cM distal to D11Mit31, and the Crybb2 locus encoding crystallin{beta}B2 maps to chromosome 5, 9.1 {+-} 4.3 cM distal to D5Mit88. The other two genes encode lens-specific gap junction and integral membrane proteins, respectively: the Gja8 locus encoding gap junction membrane channel protein {alpha}8, also called connexin50 or MP70, maps to chromosome 3, 11.9 {+-} 5.0 cM distal to D3Mit22, and the Lim2 locus encoding lens intrinsic membrane protein 2, also call MP19, maps to chromosome 7, 2.5 {+-} 2.5 cM proximal to Ngfg. All four map positions, when compared with the corresponding positions in human, lie within known regions of conserved synteny between mouse and human chromosomes. 43 refs., 2 figs., 1 tab.

  4. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  5. Plant plasma membrane protein extraction and solubilization for proteomic analysis.

    PubMed

    Santoni, Véronique

    2007-01-01

    The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.

  6. Improving membrane protein expression by optimizing integration efficiency.

    PubMed

    Niesen, Michiel J M; Marshall, Stephen S; Miller, Thomas F; Clemons, William M

    2017-09-16

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effect of double mutations, on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  7. Structures and Mechanisms of Viral Membrane Fusion Proteins

    PubMed Central

    White, Judith M.; Delos, Sue E.; Brecher, Matthew; Schornberg, Kathryn

    2009-01-01

    Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus. PMID:18568847

  8. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  9. [Plasmatic membrane protein synthesis in cells of the regenerating liver].

    PubMed

    Pospelov, A V; Gorelova, N V

    1978-05-01

    Protein synthesis in the cells of the regenerating rat liver was studied. The rate of 3H-glycine incorporation into the total proteins of the liver, those of microsomal fraction, proteins of hyaloplasm, and plasmatic membrane proteins, soluble and non-soluble in 0.05 M K2CO3, was determined. The rate of 3H-glycine incorporation into soluble proteines of plasma membranes became maximal one hour after partial hepatectomy. The peak of the rate of synthesis of proteins of other fractions fell on the end of the G1-period. A sharp increase of the synthesis rate of plasma membrane proteins seems to be one of the earliest biochemical events in the regenerating liver hepatocytes ready for division.

  10. Tuning membrane protein mobility by confinement into nanodomains

    NASA Astrophysics Data System (ADS)

    Karner, Andreas; Nimmervoll, Benedikt; Plochberger, Birgit; Klotzsch, Enrico; Horner, Andreas; Knyazev, Denis G.; Kuttner, Roland; Winkler, Klemens; Winter, Lukas; Siligan, Christine; Ollinger, Nicole; Pohl, Peter; Preiner, Johannes

    2017-03-01

    High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

  11. Fully Quantified Spectral Imaging Reveals in Vivo Membrane Protein Interactions

    PubMed Central

    King, Christopher; Stoneman, Michael; Raicu, Valerica; Hristova, Kalina

    2016-01-01

    Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling. PMID:26787445

  12. Imaging the membrane protein bacteriorhodopsin with the atomic force microscope

    SciTech Connect

    Butt, H.J.; Downing, K.H.; Hansma, P.K. )

    1990-12-01

    The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.

  13. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

    PubMed Central

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-01-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C10, was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrié et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca2+-dependent ATPase and Ca2+-pumping activity. We found that 18C10-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions. PMID:15459343

  14. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins.

    PubMed

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-11-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.

  15. Single-Molecule Microscopy and Force Spectroscopy of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Engel, Andreas; Janovjak, Harald; Fotiadis, Dimtrios; Kedrov, Alexej; Cisneros, David; Müller, Daniel J.

    Single-molecule atomic force microscopy (AFM) provides novel ways to characterize the structure-function relationship of native membrane proteins. High-resolution AFM topographs allow observing the structure of single proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. We will review these feasibilities illustrating examples of membrane proteins in native and reconstituted membranes. Classification of individual topographs of single proteins allows understanding the principles of motions of their extrinsic domains, to learn about their local structural flexibilities and to find the entropy minima of certain conformations. Combined with the visualization of functionally related conformational changes these insights allow understanding why certain flexibilities are required for the protein to function and how structurally flexible regions allow certain conformational changes. Complementary to AFM imaging, single-molecule force spectroscopy (SMFS) experiments detect molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to measure interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the locations of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes.

  16. Synaptic vesicle membrane proteins interact to form a multimeric complex

    PubMed Central

    1992-01-01

    Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal. PMID:1730776

  17. Durable vesicles for reconstitution of membrane proteins in biotechnology.

    PubMed

    Beales, Paul A; Khan, Sanobar; Muench, Stephen P; Jeuken, Lars J C

    2017-02-08

    The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. © 2017 The Author(s).

  18. Production of okara and soy protein concentrates using membrane technology.

    PubMed

    Vishwanathan, K H; Govindaraju, K; Singh, Vasudeva; Subramanian, R

    2011-01-01

    Microfiltration (MF) membranes with pore sizes of 200 and 450 nm and ultrafiltration (UF) membranes with molecular weight cut off of 50, 100, and 500 kDa were assessed for their ability to eliminate nonprotein substances from okara protein extract in a laboratory cross-flow membrane system. Both MF and UF improved the protein content of okara extract to a similar extent from approximately 68% to approximately 81% owing to the presence of protein in the feed leading to the formation of dynamic layer controlling the performance rather than the actual pore size of membranes. Although normalized flux in MF-450 (117 LMH/MPa) was close to UF-500 (118 LMH/MPa), the latter was selected based on higher average flux (47 LMH) offering the advantage of reduced processing time. Membrane processing of soy extract improved the protein content from 62% to 85% much closer to the target value. However, the final protein content in okara (approximately 80%) did not reach the target value (90%) owing to the greater presence of soluble fibers that were retained by the membrane. Solubility curve of membrane okara protein concentrate (MOPC) showed lower solubility than soy protein concentrate and a commercial isolate in the entire pH range. However, water absorption and fat-binding capacities of MOPC were either superior or comparable while emulsifying properties were in accordance with its solubility. The results of this study showed that okara protein concentrate (80%) could be produced using membrane technology without loss of any true proteins, thus offering value addition to okara, hitherto underutilized. Practical Application: Okara, a byproduct obtained during processing soybean for soymilk, is either underutilized or unutilized in spite of the fact that its protein quality is as good as that of soy milk and tofu. Membrane-processed protein products have been shown to possess superior functional properties compared to conventionally produced protein products. However, the

  19. LdFlabarin, a New BAR Domain Membrane Protein of Leishmania Flagellum

    PubMed Central

    Thonnus, Magali; Salin, Bénédicte; Boissier, Fanny; Blancard, Corinne; Sauvanet, Cécile; Metzler, Christelle; Espiau, Benoît; Sahin, Annelise; Merlin, Gilles

    2013-01-01

    During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein. PMID:24086735

  20. A bile‐inducible membrane protein mediates bifidobacterial bile resistance

    PubMed Central

    Ruiz, Lorena; O'Connell‐Motherway, Mary; Zomer, Aldert; de los Reyes‐Gavilán, Clara G.; Margolles, Abelardo; van Sinderen, Douwe

    2012-01-01

    Summary Bbr_0838 from Bifidobacterium breve UCC2003 is predicted to encode a 683 residue membrane protein, containing both a permease domain that displays similarity to transporters belonging to the major facilitator superfamily, as well as a CBS (cystathionine beta synthase) domain. The high level of similarity to bile efflux pumps from other bifidobacteria suggests a significant and general role for Bbr_0838 in bile tolerance. Bbr_0838 transcription was shown to be monocistronic and strongly induced upon exposure to bile. Further analysis delineated the transcriptional start site and the minimal region required for promoter activity and bile regulation. Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain, UCC2003:838800, which exhibited reduced survival upon cholate exposure as compared with the parent strain, a phenotype that was reversed when a functional, plasmid‐encoded Bbr_0838 gene was introduced into UCC2003:838800. Transcriptome analysis of UCC2003:838800 grown in the presence or absence of bile demonstrated that transcription of Bbr_0832, which is predicted to encode a macrolide efflux transporter gene, was significantly increased in the presence of bile, representing a likely compensatory mechanism for bile removal in the absence of Bbr_0838. This study represents the first in‐depth analysis of a bile‐inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance. PMID:22296641

  1. Dynamics of Membrane Proteins within Synthetic Polymer Membranes with Large Hydrophobic Mismatch.

    PubMed

    Itel, Fabian; Najer, Adrian; Palivan, Cornelia G; Meier, Wolfgang

    2015-06-10

    The functioning of biological membrane proteins (MPs) within synthetic block copolymer membranes is an intriguing phenomenon that is believed to offer great potential for applications in life and medical sciences and engineering. The question why biological MPs are able to function in this completely artificial environment is still unresolved by any experimental data. Here, we have analyzed the lateral diffusion properties of different sized MPs within poly(dimethylsiloxane) (PDMS)-containing amphiphilic block copolymer membranes of membrane thicknesses between 9 and 13 nm, which results in a hydrophobic mismatch between the membrane thickness and the size of the proteins of 3.3-7.1 nm (3.5-5 times). We show that the high flexibility of PDMS, which provides membrane fluidities similar to phospholipid bilayers, is the key-factor for MP incorporation.

  2. Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

    PubMed

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

  3. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    PubMed Central

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property. PMID:23383298

  4. A beta-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator.

    PubMed

    Belchik, Sara Mae; Schaeffer, Scott M; Hasenoehrl, Shelley; Xun, Luying

    2010-06-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a beta-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.

  5. A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in Cupriavidus necator

    PubMed Central

    Belchik, Sara Mae; Schaeffer, Scott M.; Hasenoehrl, Shelley

    2010-01-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds. PMID:19937267

  6. Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes.

    PubMed

    Cheng, Xi; Jo, Sunhwan; Qi, Yifei; Marassi, Francesca M; Im, Wonpil

    2015-04-21

    Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys(40), residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein

  7. Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of Xanthomonas campestris pv. Campestris

    PubMed Central

    Lee, Hsien-Ming; Wang, Kuan-Cheng; Liu, Yi-Ling; Yew, Hsin-Yan; Chen, Ling-Yun; Leu, Wei-Ming; Chen, David Chanhen; Hu, Nien-Tai

    2000-01-01

    An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of the xpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestris pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)6-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two. PMID:10692359

  8. Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

    PubMed Central

    Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

    2012-01-01

    Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

  9. Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

    PubMed

    Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

    2014-10-01

    All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

  10. Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1993-01-01

    DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies. Images PMID:8419295

  11. Overexpression of membrane proteins from higher eukaryotes in yeasts.

    PubMed

    Emmerstorfer, Anita; Wriessnegger, Tamara; Hirz, Melanie; Pichler, Harald

    2014-09-01

    Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.

  12. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices.

  13. Molecular interactions between proteins and synthetic membrane polymer films

    SciTech Connect

    Pincet, F.; Perez, E.; Belfort, G.

    1995-04-01

    To help understand the effects of protein adsorption on membrane filtration performance, we have measured the molecular interactions between cellulose acetate films and two proteins with different properties (ribonuclease A and human serum albumin) with a surface force apparatus. Comparison of forces between two protein layers with those between a protein layer and a cellulose acetate (CA) film shows that, at high pH, both proteins retained their native conformation on interacting with the CA film while at the isoelectric point (pI) or below the tertiary structure of proteins was disturbed. These measurements provide the first molecular evidence that disruption of protein tertiary structure could be responsible for the reduced permeation flows observed during membrane filtration of protein solutions and suggest that operating at high pH values away from the pI of proteins will reduce such fouling. 60 refs., 9 figs., 5 tabs.

  14. Chemical synthesis and biophysical applications of membrane proteins.

    PubMed

    Zuo, Chao; Tang, Shan; Zheng, Ji-Shen

    2015-07-01

    Chemical synthesis or semi-synthesis of membrane proteins can provide unique molecular tools, such as site-specific isotope labeling or post-translationally modified membrane proteins to gain insight into their biophysical and functional characteristics. However, during preparation, purification, and ligation of transmembrane peptides, tremendous challenges are encountered owing to their hydrophobic nature. This review focuses on the recent advances in chemical synthesis strategies of membrane proteins. These strategies help to solubilize the hydrophobic transmembrane peptide sequences under standard purification and chemical ligation conditions to improve their handling properties. Biophysical and functional studies of synthetic membrane proteins are reviewed as well. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  15. Prediction of buried helices in multispan alpha helical membrane proteins.

    PubMed

    Adamian, Larisa; Liang, Jie

    2006-04-01

    Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation. Given that the number of buried helices in a membrane protein is known, our method achieves an accuracy of 78% and a Matthew's correlation coefficient of 0.68. A server for this tool (RANTS) is available online at http://gila.bioengr.uic.edu/lab/.

  16. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

    SciTech Connect

    Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y.L.; Doniach, Sebastian; Lesley, Scott A.

    2009-10-21

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

  17. Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.

    PubMed

    Petrosyan, Rafayel; Bippes, Christian A; Walheim, Stefan; Harder, Daniel; Fotiadis, Dimitrios; Schimmel, Thomas; Alsteens, David; Müller, Daniel J

    2015-05-13

    Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

  18. Curvature forces in membrane lipid-protein interactions.

    PubMed

    Brown, Michael F

    2012-12-11

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes in membrane proteins, involving folding, stability, and membrane shape transitions, potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function.

  19. Structuring detergents for extracting and stabilizing functional membrane proteins.

    PubMed

    Matar-Merheb, Rima; Rhimi, Moez; Leydier, Antoine; Huché, Frédéric; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W; Falson, Pierre

    2011-03-31

    Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may

  20. Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins

    PubMed Central

    Matar-Merheb, Rima; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W.; Falson, Pierre

    2011-01-01

    Background Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Methodology/Principal Findings Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. Conclusion/Significance These compounds seem promising to extract in a functional state