Science.gov

Sample records for proteins putative membrane

  1. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  2. Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

    SciTech Connect

    Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

    2010-09-30

    Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

  3. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

    PubMed Central

    Gary, R; Bretscher, A

    1993-01-01

    Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8248180

  4. Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis.

    PubMed Central

    Eident-Wilkinson, B; Mele, L; Laffan, J; Firshein, W

    1992-01-01

    A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990). Images PMID:1729239

  5. Cloning, purification and preliminary crystallographic analysis of a putative DNA-binding membrane protein, YmfM, from Staphylococcus aureus

    SciTech Connect

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W.

    2008-07-01

    Truncation by the removal of the C-terminal hydrophobic transmembrane anchor has enabled the overexpression of a soluble domain of S. aureus YmfM in Escherichia coli, which has then been purified and subsequently crystallized. The Staphylococcus aureus protein YmfM contains a helix–turn–helix motif and is thought to be a putative DNA-binding protein which is associated with the membrane through a C-terminal hydrophobic transmembrane anchor. Truncation of the protein by the removal of this C-terminal hydrophobic segment has enabled the overexpression of a soluble domain of S. aureus YmfM (ΔYmfM) in Escherichia coli, which has been purified and subsequently crystallized. Crystals of ΔYmfM diffract to beyond 1.0 Å resolution and belong to one of the pair of enantiomorphic tetragonal space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 45.5, c = 72.9 Å and one molecule in the asymmetric unit. The crystals of ΔYmfM have an unusually low V{sub M} of 1.6 Å{sup 3} Da{sup −1}, which is one of the lowest values observed for any protein to date. A full structure determination is under way in order to provide insights into the function of this protein.

  6. Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.

    PubMed

    Kanoh, H; Williger, B T; Exton, J H

    1997-02-28

    ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1. PMID:9038142

  7. Monoclonal antibody against a putative myristoylated membrane protein encoded by grouper iridovirus 59L gene.

    PubMed

    Chen, Zhi-Yu; Chiou, Pinwen Peter; Liou, Chian-Jiun; Lai, Yu-Shen

    2015-04-01

    Groupers (Epinephelus spp.) are economically important fish species worldwide, and ranaviruses are major viral pathogens causing heavy economic losses in grouper aquaculture. In this study, the 59L gene of grouper iridovirus (GIV-59L) was cloned and characterized. This gene is 1521 bp and encodes a protein of 506 amino acids with a predicted molecular mass of 53.9 kDa. Interestingly, GIV-59L and its homologs are found in all genera of the family Iridoviridae. A mouse monoclonal antibody specific for the C-terminal domain (amino acid positions 254-506) of the GIV-59L protein, GIV-59L(760-1518)-MAb-21, was produced and proved to be well suited for use in a number of GIV immunoassays. RT-PCR, Western blotting, and cycloheximide and cytosine arabinoside drug inhibition analyses indicated that GIV-59L is a viral late gene in GIV-infected grouper kidney cells. Immunofluorescence analysis revealed that GIV-59L protein mainly accumulates in the cytoplasm of infected cells and is finally packed into a whole virus particle. The GIV-59L(760-1518)-MAb-21 characterized in this study could have widespread application in GIV immunodiagnostics and other research on GIV. In addition, the results presented here offer important insights into the pathogenesis of GIV. PMID:25850399

  8. Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

    PubMed

    Manni, Marco M; Sot, Jesús; Goñi, Félix M

    2015-03-01

    Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes. PMID:25485476

  9. 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

  10. Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

    PubMed Central

    Akins, D R; Robinson, E; Shevchenko, D; Elkins, C; Cox, D L; Radolf, J D

    1997-01-01

    Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function. PMID:9260949

  11. InPSR26, a Putative Membrane Protein, Regulates Programmed Cell Death during Petal Senescence in Japanese Morning Glory1[W][OA

    PubMed Central

    Shibuya, Kenichi; Yamada, Tetsuya; Suzuki, Tomoko; Shimizu, Keiichi; Ichimura, Kazuo

    2009-01-01

    The onset and progression of petal senescence, which is a type of programmed cell death (PCD), are highly regulated. Genes showing changes in expression during petal senescence in Japanese morning glory (Ipomoea nil) were isolated and examined to elucidate their function in PCD. We show here that a putative membrane protein, InPSR26, regulates progression of PCD during petal senescence in Japanese morning glory. InPSR26 is dominantly expressed in petal limbs and its transcript level increases prior to visible senescence symptoms. Transgenic plants with reduced InPSR26 expression (PSR26r lines) showed accelerated petal wilting, with PCD symptoms including cell collapse, ion and anthocyanin leakage, and DNA degradation accelerated in petals compared to wild-type plants. Transcript levels of autophagy- and PCD-related genes (InATG4, InATG8, InVPE, and InBI-1) were reduced in the petals of PSR26r plants. Autophagy visualized by monodansylcadaverine staining confirmed that autophagy is induced in senescing petal cells of wild-type plants and that the percentage of cells containing monodansylcadaverine-stained structures, most likely autophagosomes, was significantly lower in the petals of PSR26r plants, indicating reduced autophagic activity in the PSR26r plants. These results suggest that InPSR26 acts to delay the progression of PCD during petal senescence, possibly through regulation of the autophagic process. Our data also suggest that autophagy delays PCD in petal senescence. PMID:19036837

  12. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    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 cells 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 of 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

  13. Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

    PubMed

    Hashimoto, Masayoshi; Komatsu, Ken; Iwai, Ryo; Keima, Takuya; Maejima, Kensaku; Shiraishi, Takuya; Ishikawa, Kazuya; Yoshida, Tetsuya; Kitazawa, Yugo; Okano, Yukari; Yamaji, Yasuyuki; Namba, Shigetou

    2015-06-01

    Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses. PMID:25650831

  14. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  15. Lipid membranes for membrane proteins.

    PubMed

    Kukol, Andreas

    2015-01-01

    The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile. PMID:25330959

  16. 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

  17. 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

  18. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  19. Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin

    PubMed Central

    Perlińska, Agata

    2014-01-01

    Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche. PMID:25426338

  20. Putative pore-loops of TMEM16/anoctamin channels affect channel density in cell membranes.

    PubMed

    Adomaviciene, Aiste; Smith, Keith J; Garnett, Hannah; Tammaro, Paolo

    2013-07-15

    The recently identified TMEM16/anoctamin protein family includes Ca(2+)-activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca(2+) binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca(2+). Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca(2+) or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore-loop of TMEM16A and TMEM16B channels in the control of the whole-cell Ca(2+)-activated Cl(-) conductance. PMID:23613533

  1. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  2. Membrane Protein Prediction Methods

    PubMed Central

    Punta, Marco; Forrest, Lucy R.; Bigelow, Henry; Kernytsky, Andrew; Liu, Jinfeng; Rost, Burkhard

    2007-01-01

    We survey computational approaches that tackle membrane protein structure and function prediction. While describing the main ideas that have led to the development of the most relevant and novel methods, we also discuss pitfalls, provide practical hints and highlight the challenges that remain. The methods covered include: sequence alignment, motif search, functional residue identification, transmembrane segment and protein topology predictions, homology and ab initio modeling. Overall, predictions of functional and structural features of membrane proteins are improving, although progress is hampered by the limited amount of high-resolution experimental information available. While predictions of transmembrane segments and protein topology rank among the most accurate methods in computational biology, more attention and effort will be required in the future to ameliorate database search, homology and ab initio modeling. PMID:17367718

  3. Membrane Bending by Protein Crowding

    NASA Astrophysics Data System (ADS)

    Stachowiak, Jeanne

    2014-03-01

    From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

  4. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

    PubMed Central

    Soni, Siddarth; Raaijmakers, Antonia J. A.; Raaijmakers, Linsey M.; Damen, J. Mirjam A.; van Stuijvenberg, Leonie; Vos, Marc A.; Heck, Albert J. R.

    2016-01-01

    Aims Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue. Methods and Results General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes. Conclusion The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart. PMID:27148881

  5. Tracking Membrane Protein Association in Model Membranes

    PubMed Central

    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 Å, 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

  6. 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. PMID:26360742

  7. Microtechnologies for membrane protein studies

    PubMed Central

    Suzuki, Hiroaki

    2008-01-01

    Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research. PMID:18335213

  8. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    SciTech Connect

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  9. 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.

  10. Putative Mineral-Specific Proteins Synthesized by a Metal Reducing Bacterium

    SciTech Connect

    Lower, Brian H.; Hochella Jr., Michael F.; Lower, Steven K.

    2006-02-01

    Biological force microscopy (BFM) was combined with two-dimensional (2D) gel electrophoresis and mass spectrometry to identify outer membrane proteins (OM) from Shewanella oneidensis that are involved in anaerobic Fe(III) reduction. This is the first time that biophysical force measurements have been coupled with protein expression patterns to search for evidence of putative mineral-specific proteins synthesized by bacteria. BFM shows that S. oneidensis possess an affinity towards goethite (FeOOH) but not diaspore (AlOOH) under anaerobic conditions, despite the fact that diaspore is isostructural with goethite and has essentially the same surface charge. The worm-like chain model was used to identify force-signatures in BFM-derived force curves indicative of putative outer membrane (OM) polypeptides synthesized by S. oneidensis to form a bond with goethite. Protein expression patterns from OM extract of cells grown under anaerobic Fe(III) reducing versus aerobic conditions show that approximately 400 protein spots exhibit significant differences in abundance on 2D gels. Peptide mass fingerprinting and tandem mass spectrometry were used to identify several of the protein spots that were significantly more abundant in 2D gels from OM extract of cells grown under anaerobic Fe(III) reducing conditions. Among those identified were proteins involved in Fe(III) and Mn(IV) reduction, protein transport and secretion, polysaccharide biosynthesis and export, and hypothetical proteins with unknown functions. Together, the BFM and proteomic data suggest that OM proteins are synthesized by S. oneidensis under anaerobic conditions to function in iron oxide binding and/or Fe(III) reduction. If this is the case, then it is possible that the evolution of dissimilatory iron-reducing bacteria like Shewanella, could have been, at least in part, driven by the binding/reduction ability of certain proteins to specific mineral phases.

  11. Membrane Protein Assembly into Nanodiscs

    PubMed Central

    Bayburt, Timothy H.; Sligar, Stephen G.

    2016-01-01

    Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins. PMID:19836392

  12. Structure Prediction of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Hu, Xiche

    Membrane proteins play a central role in many cellular and physiological processes. It is estimated that integral membrane proteins make up about 20-30% of the proteome (Krogh et al., 2001b; Stevens and Arkin, 2000; von Heijne, 1999). They are essential mediators of material and information transfer across cell membranes. Their functions include active and passive transport of molecules into and out of cells and organelles; transduction of energy among various forms (light, electrical, and chemical energy); as well as reception and transduction of chemical and electrical signals across membranes (Avdonin, 2005; Bockaert et al., 2002; Pahl, 1999; Rehling et al., 2004; Stack et al., 1995). Identifying these transmembrane (TM) proteins and deciphering their molecular mechanisms, then, is of great importance, particularly as applied to biomedicine. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances, and are directly involved in their uptake, metabolism, and clearance (Bettler et al., 1998; Cohen, 2002; Heusser and Jardieu, 1997; Tibes et al., 2005; Xu et al., 2005). Despite the importance of membrane proteins, the knowledge of their high-resolution structures and mechanisms of action has lagged far behind in comparison to that of water-soluble proteins: less than 1% of all three-dimensional structures deposited in the Protein Data Bank are of membrane proteins. This unfortunate disparity stems from difficulties in overexpression and the crystallization of membrane proteins (Grisshammer and Tate, 1995; Michel, 1991).

  13. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    PubMed

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  14. A comprehensive strategy to identify stoichiometric membrane protein interactomes

    PubMed Central

    Gokhale, Avanti; Perez-Cornejo, Patricia; Duran, Charity; Hartzell, H. Criss; Faundez, Victor

    2012-01-01

    There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies. PMID:23676845

  15. 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.

  16. HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

    PubMed Central

    Hopfe, Miriam; Hegemann, Johannes H; Henrich, Birgit

    2005-01-01

    Background HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. Results An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase

  17. 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

  18. 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.

  19. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGESBeta

    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

  20. Identification of a novel putative mitochondrial protein FAM210B associated with erythroid differentiation.

    PubMed

    Kondo, Aiko; Fujiwara, Tohru; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Nakamura, Yukio; Sawada, Kenichi; Harigae, Hideo

    2016-04-01

    The transcription factor GATA-1 plays an essential role in erythroid differentiation. To identify novel GATA-1 target genes, we analyzed a merged ChIP-seq and expression profiling dataset. We identified FAM210B as a putative novel GATA-1 target gene. Study results demonstrated that GATA-1 directly regulates FAM210B expression, presumably by binding to an intronic enhancer region. Both human and murine FAM210B are abundantly expressed in the later stages of erythroblast development. Moreover, the deduced amino acid sequence predicted that FAM210B is a membrane protein, and Western blot analysis demonstrated its mitochondrial localization. Loss-of-function analysis in erythroid cells suggested that FAM210B may be involved in erythroid differentiation. The identification and characterization of FAM210B provides new insights in the study of erythropoiesis and hereditary anemias. PMID:26968549

  1. Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

    PubMed Central

    Xu, Jinbin; Zeng, Chenbo; Chu, Wenhua; Pan, Fenghui; Rothfuss, Justin M.; Zhang, Fanjie; Tu, Zhude; Zhou, Dong; Zeng, Dexing; Vangveravong, Suwanna; Johnston, Fabian; Spitzer, Dirk; Chang, Katherine C.; Hotchkiss, Richard S.; Hawkins, William G.; Wheeler, Kenneth T.; Mach, Robert H.

    2013-01-01

    The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumor cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor binding site. WC-21, a sigma-2 ligand containing both a photoactive moiety azide and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was then identified as PGRMC1 (progesterone receptor membrane component-1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalizes with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex. PMID:21730960

  2. Multiscale Simulation of Protein Mediated Membrane Remodeling

    PubMed Central

    Ayton, Gary S.; Voth, Gregory A.

    2009-01-01

    Proteins interacting with membranes can result in substantial membrane deformations and curvatures. This effect is known in its broadest terms as membrane remodeling. This review article will survey current multiscale simulation methodologies that have been employed to examine protein-mediated membrane remodeling. PMID:19922811

  3. Identification of a putative yeast homolog of the mammalian beta chains of the clathrin-associated protein complexes.

    PubMed Central

    Kirchhausen, T

    1990-01-01

    The clathrin-associated protein complexes are heterotetrameric structures believed to interact with clathrin and with membrane components of mammalian coated pits and coated vesicles. I have identified a yeast homolog of the mammalian beta-type large chains, suggesting the existence in yeast cells of clathrin-associated protein complexes. A sequence comparison between the putative yeast beta-type chain and its mammalian counterparts shows that their amino-terminal domains are related over their entire length and that their carboxyl-terminal domains diverge completely. This observation is consistent with our earlier proposal (T. Kurchhausen et al., Proc. Natl. Acad. Sci. USA 86:2612-2616, 1989) for the bifunctional-domain organization of the large chains, in which the invariant amino-terminal region interacts with conserved proteins of the coat while the variable carboxyl-terminal domain interacts with different membrane components of coated pits and coated vesicles. PMID:2122239

  4. An Arg-rich putative prebiotic protein is as stable as its Lys-rich variant.

    PubMed

    Diez-García, Fernando; Chakrabartty, Avijit; González, Carlos; Laurents, Douglas V

    2012-12-15

    An Arg-rich peptide called RIA7; sequence ac-ARAAAAAIRAIAAIIRAGGY-am, tetramerizes to form a well folded, four helix X-bundle protein. The Arg side chains are solvent exposed and the hydrophobic core is composed of the side chains from some Alas, all the Iles and the C-terminal Tyr. Since Gly, Ala and Ile, and in lesser amounts Arg and Tyr have been reported to form under putative prebiotic Earth conditions, it is plausible that RIA7-like peptides might have formed on the primitive Earth and interacted with RNAs. The interaction of RIA7 with two RNAs was tested and the formation of insoluble aggregates was observed. These results contrast with previous studies of a Lys-rich variant, called KIA7, which promotes the cleavage of RNAs. Their close structural similarity makes RIA7 and KIA7 an excellent system to compare the relative contributions of Arg and Lys to protein conformational stability. NMR-monitored hydrogen/deuterium exchange measurements and CD-monitored thermal denaturation experiments performed at different peptide and salt concentrations reveal that the conformational stabilities of RIA7 and KIA7 are practically the same. This finding has relevance for protein engineering as Lys is frequently replaced by Arg to improve ligand binding and membrane association and penetration. PMID:23022061

  5. Dendrotoxin acceptor from bovine synaptic plasma membranes. Binding properties, purification and subunit composition of a putative constituent of certain voltage-activated K+ channels.

    PubMed Central

    Parcej, D N; Dolly, J O

    1989-01-01

    Dendrotoxin is a snake polypeptide that blocks selectively and potently certain voltage-sensitive, fast-activating K+ channels in the nervous system, where it binds with high affinity to membranous acceptors. Herein, the acceptor protein for dendrotoxin in bovine synaptic membranes is solubilized in active form and its complete purification achieved by affinity chromatography, involving a novel elution procedure. This putative K+-channel constituent is shown to be a large oligomeric glycoprotein containing two major subunits, with Mr values of 75,000 and 37,000. Images Fig. 2. PMID:2930493

  6. Influences of Membrane Mimetic Environments on Membrane Protein Structures

    PubMed Central

    Zhou, Huan-Xiang; Cross, Timothy A.

    2013-01-01

    The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins. PMID:23451886

  7. 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

  8. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  9. 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.

  10. Mso1p regulates membrane fusion through interactions with the putative N-peptide-binding area in Sec1p domain 1.

    PubMed

    Weber, Marion; Chernov, Konstantin; Turakainen, Hilkka; Wohlfahrt, Gerd; Pajunen, Maria; Savilahti, Harri; Jäntti, Jussi

    2010-04-15

    Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p-Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion. PMID:20181830

  11. Mso1p Regulates Membrane Fusion through Interactions with the Putative N-Peptide–binding Area in Sec1p Domain 1

    PubMed Central

    Weber, Marion; Chernov, Konstantin; Turakainen, Hilkka; Wohlfahrt, Gerd; Pajunen, Maria; Savilahti, Harri

    2010-01-01

    Sec1p/Munc18 (SM) family proteins regulate SNARE complex function in membrane fusion through their interactions with syntaxins. In addition to syntaxins, only a few SM protein interacting proteins are known and typically, their binding modes with SM proteins are poorly characterized. We previously identified Mso1p as a Sec1p-binding protein and showed that it is involved in membrane fusion regulation. Here we demonstrate that Mso1p and Sec1p interact at sites of exocytosis and that the Mso1p–Sec1p interaction site depends on a functional Rab GTPase Sec4p and its GEF Sec2p. Random and targeted mutagenesis of Sec1p, followed by analysis of protein interactions, indicates that Mso1p interacts with Sec1p domain 1 and that this interaction is important for membrane fusion. In many SM family proteins, domain 1 binds to a N-terminal peptide of a syntaxin family protein. The Sec1p-interacting syntaxins Sso1p and Sso2p lack the N-terminal peptide. We show that the putative N-peptide binding area in Sec1p domain 1 is important for Mso1p binding, and that Mso1p can interact with Sso1p and Sso2p. Our results suggest that Mso1p mimics N-peptide binding to facilitate membrane fusion. PMID:20181830

  12. Desmin: molecular interactions and putative functions of the muscle intermediate filament protein.

    PubMed

    Costa, M L; Escaleira, R; Cataldo, A; Oliveira, F; Mermelstein, C S

    2004-12-01

    Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions. PMID:15558188

  13. Membrane proteins: always an insoluble problem?

    PubMed

    Rawlings, Andrea E

    2016-06-15

    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. 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

  15. Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

    PubMed

    Kim, Kyung-mi; Adyshev, Djanybek M; Kása, Anita; Zemskov, Evgeny A; Kolosova, Irina A; Csortos, Csilla; Verin, Alexander D

    2013-07-01

    We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells. PMID:23583905

  16. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

    PubMed

    Dubytska, Lidiya P; Rogge, Matthew L; Thune, Ronald L

    2016-01-01

    Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  17. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    ABSTRACT Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

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

    PubMed Central

    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. PMID:25569184

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

    PubMed

    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. PMID:25569184

  20. 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.

  1. Cloning of a Membrane Protein that Induces a Slow Voltage-Gated Potassium Current

    NASA Astrophysics Data System (ADS)

    Takumi, Toru; Ohkubo, Hiroaki; Nakanishi, Shigetada

    1988-11-01

    A rat kidney messenger RNA that induces a slowly activating, voltage-dependent potassium current on its expression in Xenopus oocytes was identified by combining molecular cloning with an electrophysiological assay. The cloned complementary DNA encodes a novel membrane protein that consists of 130 amino acids with a single putative transmembrane domain. This protein differs from the known ion channel proteins but is involved in the induction of selective permeation of potassium ions by membrane depolarization.

  2. AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes1[w

    PubMed Central

    Reumann, Sigrun; Ma, Changle; Lemke, Steffen; Babujee, Lavanya

    2004-01-01

    To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783–800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in β-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid α-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes. PMID:15333753

  3. 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

  4. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  5. 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

  6. Solid state NMR and protein-protein interactions in membranes.

    PubMed

    Miao, Yimin; Cross, Timothy A

    2013-12-01

    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

  7. 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.

  8. 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. PMID:27284055

  9. 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

  10. Crystal Dehydration in Membrane Protein Crystallography.

    PubMed

    Sanchez-Weatherby, Juan; Moraes, Isabel

    2016-01-01

    Crystal dehydration has been successfully implemented to facilitate the structural solution of a number of soluble and membrane protein structures over the years. This chapter will present the currently available tools to undertake controlled crystal dehydration, focusing on some successful membrane protein cases. Also discussed here will be some practical considerations regarding membrane protein crystals and the relationship between different techniques in order to help researchers to select the most suitable technique for their projects. PMID:27553236

  11. Putative signal peptides of two BURP proteins can direct proteins to their destinations in tobacco cell system.

    PubMed

    Tang, Yulin; Ou, Zhonghua; Qiu, Jianbin; Mi, Zilan

    2014-11-01

    Plant-specific BURP family proteins have a diverse subcellular localization with different functions. However, only limited studies have investigated the functions of their different domains. In the present study, the role of the N-terminal putative signal peptide in protein subcellular localization was investigated using a tobacco cell system. The results showed that SALI3-2 was present in vacuoles, whereas AtRD22 was directed to the apoplast. The N-terminal putative signal peptides of both proteins were confirmed to be the essential and critical domains for targeting the proteins to their destinations. We also demonstrate that the expression and accumulation of mGFP in tobacco cells was increased when mGFP was fused to the putative signal peptide of SALI3-2. The findings offer the potential application of this short peptide in protein production in plants. PMID:25048229

  12. Return to sender: use of Plasmodium ER retrieval sequences to study protein transport in the infected erythrocyte and predict putative ER protein families.

    PubMed

    Külzer, Simone; Gehde, Nina; Przyborski, Jude M

    2009-06-01

    We have investigated how knowledge of endoplasmic reticulum (ER) retrieval signals can be used to study specific trafficking pathways in the malaria-infected erythrocyte. We show that addition of various lumenal ER retrieval signals to soluble green fluorescent protein (GFP) chimaera causes retrieval of the fusion protein in the parasite's ER. In contrast, adding these signals to the C-terminus of a membrane bound protein does not affect its eventual sub-cellular localization. This demonstrates proof of principle that ER retrieval signals can be used to study the solubility state of Plasmodium falciparum proteins during their transport to the host erythrocyte. Furthermore, using our knowledge of ER retrieval signals, we identify Plasmodium ER protein families and assign putative functions to them. PMID:19294420

  13. Role for Chlamydial Inclusion Membrane Proteins in Inclusion Membrane Structure and Biogenesis

    PubMed Central

    Mital, Jeffrey; Miller, Natalie J.; Dorward, David W.; Dooley, Cheryl A.; Hackstadt, Ted

    2013-01-01

    The chlamydial inclusion membrane is extensively modified by the insertion of type III secreted effector proteins. These inclusion membrane proteins (Incs) are exposed to the cytosol and share a common structural feature of a long, bi-lobed hydrophobic domain but little or no primary amino acid sequence similarity. Based upon secondary structural predictions, over 50 putative inclusion membrane proteins have been identified in Chlamydia trachomatis. Only a limited number of biological functions have been defined and these are not shared between chlamydial species. Here we have ectopically expressed several C. trachomatis Incs in HeLa cells and find that they induce the formation of morphologically distinct membranous vesicular compartments. Formation of these vesicles requires the bi-lobed hydrophobic domain as a minimum. No markers for various cellular organelles were observed in association with these vesicles. Lipid probes were incorporated by the Inc-induced vesicles although the lipids incorporated were dependent upon the specific Inc expressed. Co-expression of Inc pairs indicated that some colocalized in the same vesicle, others partially overlapped, and others did not associate at all. Overall, it appears that Incs may have an intrinsic ability to induce membrane formation and that individual Incs can induce membranous structures with unique properties. PMID:23696825

  14. Serial Femtosecond Crystallography of Membrane Proteins.

    PubMed

    Zhu, Lan; Weierstall, Uwe; Cherezov, Vadim; Liu, Wei

    2016-01-01

    Membrane proteins, including G protein-coupled receptors (GPCRs), constitute the most important drug targets. The increasing number of targets requires new structural information, which has proven tremendously challenging due to the difficulties in growing diffraction-quality crystals. Recent developments of serial femtosecond crystallography at X-ray free electron lasers combined with the use of membrane-mimetic gel-like matrix of lipidic cubic phase (LCP-SFX) for crystal growth and delivery hold significant promise to accelerate structural studies of membrane proteins. This chapter describes the development and current status of the LCP-SFX technology and elaborates its future role in structural biology of membrane proteins. PMID:27553241

  15. Class II virus membrane fusion proteins

    SciTech Connect

    Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2006-01-05

    Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses.

  16. Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

    PubMed Central

    Molitoris, B A; Wilson, P D; Schrier, R W; Simon, F R

    1985-01-01

    and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores. Images PMID:3001141

  17. Mapping membrane protein structure with fluorescence

    PubMed Central

    Taraska, Justin W.

    2012-01-01

    Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer, and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes. PMID:22445227

  18. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  19. Membrane Protein Insertion at the Endoplasmic Reticulum

    PubMed Central

    Shao, Sichen; Hegde, Ramanujan S.

    2014-01-01

    Integral membrane proteins of the cell surface and most intracellular compartments of eukaryotic cells are assembled at the endoplasmic reticulum. Two highly conserved and parallel pathways mediate membrane protein targeting to and insertion into this organelle. The classical cotranslational pathway, utilized by most membrane proteins, involves targeting by the signal recognition particle followed by insertion via the Sec61 translocon. A more specialized posttranslational pathway, employed by many tail-anchored membrane proteins, is composed of entirely different factors centered around a cytosolic ATPase termed TRC40 or Get3. Both of these pathways overcome the same biophysical challenges of ferrying hydrophobic cargo through an aqueous milieu, selectively delivering it to one among several intracellular membranes and asymmetrically integrating its transmembrane domain(s) into the lipid bilayer. Here, we review the conceptual and mechanistic themes underlying these core membrane protein insertion pathways, the complexities that challenge our understanding, and future directions to over-come these obstacles. PMID:21801011

  20. Unlocking the eukaryotic membrane protein structural proteome

    PubMed Central

    Lee, John Kyongwon; Stroud, Robert Michael

    2012-01-01

    Summary Most of the 231 unique membrane protein structures (as of 3/2010) are of bacterial membrane proteins (MPs) expressed in bacteria, or eukaryotic MPs from natural sources. However eukaryotic membrane proteins, especially those with more than three membrane crossings rarely succumb to any suitable expression in bacterial cells. They typically require expression in eukaryotic cells that can provide appropriate endoplasmic reticulum, chaperones, targeting and post-translational processing. In evidence, only ~20 eukaryotic MP structures have resulted from heterologous expression. This is required for a general approach to target particular human or pathogen membrane proteins of importance to human health. The first of these appeared in 2005. Our review addresses the special issues that pertain to the expression of eukaryotic and human membrane proteins, and recent advances in the tool kit for crystallization and structure determination. PMID:20739007

  1. Functional characterization of a putative disaccharide membrane transporter in crustacean intestine.

    PubMed

    Likely, Rasheda; Johnson, Eric; Ahearn, Gregory A

    2015-02-01

    Transepithelial absorption of dietary sucrose in the American lobster, Homarus americanus, was investigated by mounting an intestine in a perfusion chamber to characterize mucosal to serosal (MS) (14)C-sucrose transport. These fluxes were measured by adding varying concentrations of (14)C-sucrose to the perfusate and monitoring their appearance in the bathing solution. Transepithelial (14)C-sucrose transport was the combination of a hyperbolic function of luminal concentration, following Michaelis-Menten kinetics, and apparent diffusion. The kinetic constants of the putative sucrose transporter were KM = 20.50 ± 6.00 µM and J max = 1.81 ± 0.50 pmol/cm(2) × min. Phloridzin, an inhibitor of Na(+)-dependent mucosal glucose transport, decreased MS (14)C-sucrose transport. Decreased MS (14)C-sucrose transport also occurred in the presence of luminal trehalose, a disaccharide containing D-glucose moieties. Thin-layer chromatography (TLC) identified the chemical nature of radioactively labeled sugars in the bath following transepithelial transport. TLC revealed (14)C-sucrose was transported across the intestine largely intact with no (14)C-glucose or (14)C-fructose appearing in the serosal bath or luminal perfusate. Only 13% of bath radioactivity was volatile metabolites. Results suggest that disaccharide sugars can be transported intact across crustacean intestine and support the occurrence of a functional disaccharide membrane transporter. PMID:25416426

  2. Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

    PubMed Central

    Campos, Cristine G.; Borst, Luke

    2013-01-01

    Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen. PMID:23340315

  3. Circular dichroism spectroscopy of membrane proteins.

    PubMed

    Miles, A J; Wallace, B A

    2016-09-21

    Circular dichroism (CD) spectroscopy is a well-established technique for studying the secondary structures, dynamics, folding pathways, and interactions of soluble proteins, and is complementary to the high resolution but generally static structures produced by X-ray crystallography, NMR spectroscopy, and cryo electron microscopy. CD spectroscopy has special relevance for the study of membrane proteins, which are difficult to crystallise and largely ignored in structural genomics projects. However, the requirement for membrane proteins to be embedded in amphipathic environments such as membranes, lipid vesicles, detergent micelles, bicelles, oriented bilayers, or nanodiscs, in order for them to be soluble or dispersed in solution whilst maintaining their structure and function, necessitates the use of different experimental and analytical approaches than those employed for soluble proteins. This review discusses specialised methods for collecting and analysing membrane protein CD data, highlighting where protocols for soluble and membrane proteins diverge. PMID:27347568

  4. Phosphoinositide Control of Membrane Protein Function

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K.; Cui, Meng; Baki, Lia

    2015-01-01

    Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526

  5. Effects of protein crowding on membrane systems.

    PubMed

    Guigas, Gernot; Weiss, Matthias

    2016-10-01

    Cellular membranes are typically decorated with a plethora of embedded and adsorbed macromolecules, e.g. proteins, that participate in numerous vital processes. With typical surface densities of 30,000 proteins per μm(2) cellular membranes are indeed crowded places that leave only few nanometers of private space for individual proteins. Here, we review recent advances in our understanding of protein crowding in membrane systems. We first give a brief overview on state-of-the-art approaches in experiment and simulation that are frequently used to study crowded membranes. After that, we review how crowding can affect diffusive transport of proteins and lipids in membrane systems. Next, we discuss lipid and protein sorting in crowded membrane systems, including effects like protein cluster formation, phase segregation, and lipid droplet formation. Subsequently, we highlight recent progress in uncovering crowding-induced conformational changes of membranes, e.g. membrane budding and vesicle formation. Finally, we give a short outlook on potential future developments in the field of crowded membrane systems. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26724385

  6. Structure of the HIV-1 gp41 Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation

    SciTech Connect

    Liu, J.; Deng, Y; Dey, A; Moore, J; Lu, M

    2009-01-01

    The conserved membrane-proximal external region (MPER) of the HIV-1 gp41 envelope protein is the established target for very rare but broadly neutralizing monoclonal antibodies (NAbs) elicited during natural human infection. Nevertheless, attempts to generate an HIV-1 neutralizing antibody response with immunogens bearing MPER epitopes have met with limited success. Here we show that the MPER peptide (residues 662-683) forms a labile ?-helical trimer in aqueous solution and report the crystal structure of this autonomous folding subdomain stabilized by addition of a C-terminal isoleucine zipper motif. The structure reveals a parallel triple-stranded coiled coil in which the neutralization epitope residues are buried within the interface between the associating MPER helices. Accordingly, both the 2F5 and 4E10 NAbs recognize the isolated MPER peptide but fail to bind the trimeric MPER subdomain. We propose that the trimeric MPER structure represents the prefusion conformation of gp41, preceding the putative prehairpin intermediate and the postfusion trimer-of-hairpins structure. As such, the MPER trimer should inform the design of new HIV-1 immunogens to elicit broadly neutralizing antibodies.

  7. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    PubMed Central

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer; Miercke, Larry J. W.; Rasmussen, Søren G. F.; Robbins, Rebecca A.; Rana, Rohini R.; Loland, Claus J.; Kobilka, Brian; Stroud, Robert; Byrne, Bernadette; Gether, Ulrik; Gellman, Samuel H.

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles. PMID:21049926

  8. Vacuole membrane protein 1, autophagy and much more.

    PubMed

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Escalante, Ricardo

    2008-08-01

    Vacuole membrane protein 1 (Vmp1) is a putative transmembrane protein that has been associated with different functions including autophagy, cell adhesion, and membrane traffic. Highly similar proteins are present in lower eukaryotes and plants although a homologue is absent in the fungi lineage. We have recently described the first loss-of-function mutation for a Vmp1 homologue in a model system, Dictyostelium discoideum. Our results give a more comprehensive view of the intricate roles played by this new gene. Dictyostelium Vmp1 is an endoplasmic reticulum-resident protein. Cells deficient in Vmp1 display pleiotropic defects in the context of the secretory pathway such as organelle biogenesis, the endocytic pathway, and protein secretion. The biogenesis of the contractile vacuole, an organelle necessary to survive under hypoosmotic conditions, is compromised as well as the structure of the endoplasmic reticulum and the Golgi apparatus. Transmission electron microscopy also shows abnormal accumulation of aberrant double-membrane vesicles, suggesting a defect in autophagosome biogenesis or maturation. The expression of a mammalian Vmp1 in the Dictyostelium mutant complements the phenotype suggesting a functional conservation during evolution. We are taking the first steps in understanding the function of this fascinating protein and recent studies have brought us more questions than answers about its basic function and its role in human pathology. PMID:18641456

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Genome-wide Membrane Protein Structure Prediction

    PubMed Central

    Piccoli, Stefano; Suku, Eda; Garonzi, Marianna; Giorgetti, Alejandro

    2013-01-01

    Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome. PMID:24403851

  14. Multipass Membrane Protein Structure Prediction Using Rosetta

    PubMed Central

    Yarov-Yarovoy, Vladimir; Schonbrun, Jack; Baker, David

    2006-01-01

    We describe the adaptation of the Rosetta de novo structure prediction method for prediction of helical transmembrane protein structures. The membrane environment is modeled by embedding the protein chain into a model membrane represented by parallel planes defining hydrophobic, interface, and polar membrane layers for each energy evaluation. The optimal embedding is determined by maximizing the exposure of surface hydrophobic residues within the membrane and minimizing hydrophobic exposure outside of the membrane. Protein conformations are built up using the Rosetta fragment assembly method and evaluated using a new membrane-specific version of the Rosetta low-resolution energy function in which residue–residue and residue–environment interactions are functions of the membrane layer in addition to amino acid identity, distance, and density. We find that lower energy and more native-like structures are achieved by sequential addition of helices to a growing chain, which may mimic some aspects of helical protein biogenesis after translocation, rather than folding the whole chain simultaneously as in the Rosetta soluble protein prediction method. In tests on 12 membrane proteins for which the structure is known, between 51 and 145 residues were predicted with root-mean-square deviation <4Å from the native structure. PMID:16372357

  15. 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.

  16. Inherently Tunable Electrostatic Assembly of Membrane Proteins

    SciTech Connect

    Liang, H.; Whited, G.; Nguyen, C.; Okerlund, A.; Stucky, G.D.

    2009-05-19

    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.

  17. In Silico Analysis of Putative Paralytic Shellfish Poisoning Toxins Export Proteins in Cyanobacteria

    PubMed Central

    Soto-Liebe, Katia; López-Cortés, Xaviera A.; Fuentes-Valdes, Juan José; Stucken, Karina; Gonzalez-Nilo, Fernando; Vásquez, Mónica

    2013-01-01

    Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na+ channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H+) or sodium (Na+) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na+ and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na+ recognition domain was conserved in both SxtF/M, indicating that Na+ can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na+ incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na+ in toxin export, as well as the motifs L398XGLQD

  18. 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

  19. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    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

  20. 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

  1. Glasslike Membrane Protein Diffusion in a Crowded Membrane.

    PubMed

    Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

    2016-02-23

    Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion. PMID:26859708

  2. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

  3. Polyene antibiotic that inhibits membrane transport proteins

    PubMed Central

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

    2012-01-01

    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. PMID:22733749

  4. 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. PMID:22733749

  5. 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

  6. Biophysical EPR Studies Applied to Membrane Proteins

    PubMed Central

    Sahu, Indra D; Lorigan, Gary A

    2015-01-01

    Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented. PMID:26855825

  7. Towards Simulations of Outer Membrane Proteins in Lipopolysaccharide Membranes

    SciTech Connect

    Soares, Thereza A.; Straatsma, TP

    2007-12-26

    Biomolecular simulation derived properties of LPS membranes that impact the structural and internal dynamics of transmembrane proteins are shown to exhibit good agreement with available experimental data within the time scale simulated, chosen force field and simulation conditions. The molecular model used offers an accurate representation of the LPS layer, including the high asymmetry and low fluidity characteristics of outer membranes. This contribution describes the data intensive analysis of the large molecular time trajectories generated for these systems using massively parallel computing resources.

  8. Membrane proteins: is the future disc shaped?

    PubMed

    Lee, Sarah C; Pollock, Naomi L

    2016-08-15

    The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology. PMID:27528746

  9. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  10. 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. PMID:27553232

  11. 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

  12. Detergent-Free Membrane Protein Purification.

    PubMed

    Rothnie, Alice J

    2016-01-01

    Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment. PMID:27485341

  13. The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

    PubMed Central

    Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

    2015-01-01

    Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

  14. Dielectrophoretic Sorting of Membrane Protein Nanocrystals

    PubMed Central

    Abdallah, Bahige G.; Chao, Tzu-Chiao; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2013-01-01

    Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography. PMID:24004002

  15. Efficient preparation and analysis of membrane and membrane protein systems.

    PubMed

    Javanainen, Matti; Martinez-Seara, Hector

    2016-10-01

    Molecular dynamics (MD) simulations have become a highly important technique to consider lipid membrane systems, and quite often they provide considerable added value to laboratory experiments. Rapid development of both software and hardware has enabled the increase of time and size scales reachable by MD simulations to match those attainable by several accurate experimental techniques. However, until recently, the quality and maturity of software tools available for building membrane models for simulations as well as analyzing the results of these simulations have seriously lagged behind. Here, we discuss the recent developments of such tools from the end-users' point of view. In particular, we review the software that can be employed to build lipid bilayers and other related structures with or without embedded membrane proteins to be employed in MD simulations. Additionally, we provide a brief critical insight into force fields and MD packages commonly used for membrane and membrane protein simulations. Finally, we list analysis tools that can be used to study the properties of membrane and membrane protein systems. In all these points we comment on the respective compatibility of the covered tools. We also share our opinion on the current state of the available software. We briefly discuss the most commonly employed tools and platforms on which new software can be built. We conclude the review by providing a few ideas and guidelines on how the development of tools can be further boosted to catch up with the rapid pace at which the field of membrane simulation progresses. This includes improving the compatibility between software tools and promoting the openness of the codes on which these applications rely. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26947184

  16. NMR of Membrane Proteins: Beyond Crystals.

    PubMed

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  17. Surface recognition elements of membrane protein oligomerization.

    PubMed

    Rath, Arianna; Deber, Charles M

    2008-02-15

    Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts. PMID:17729275

  18. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:26139252

  19. Biophysical Characterization of Membrane Proteins in Nanodiscs

    PubMed Central

    Inagaki, Sayaka; Ghirlando, Rodolfo; Grisshammer, Reinhard

    2012-01-01

    Nanodiscs are self-assembled discoidal phospholipid bilayers surrounded and stabilized by membrane scaffold proteins (MSP), that have become a powerful and promising tool for the study of membrane proteins. Even though their reconstitution is highly regulated by the type of MSP and phospholipid input, a biophysical characterization leading to the determination of the stoichiometry of MSP, lipid and membrane protein is essential. This is important for biological studies, as the oligomeric state of membrane proteins often correlates with their functional activity. Typically combinations of several methods are applied using, for example, modified samples that incorporate fluorescent labels, along with procedures that result in nanodisc disassembly and lipid dissolution. To obtain a comprehensive understanding of the native properties of nanodiscs, modification-free analysis methods are required. In this work we provide a strategy, using a combination of dynamic light scattering and analytical ultracentrifugation, for the biophysical characterization of unmodified nanodiscs. In this manner we characterize the nanodisc preparation in terms of its overall polydispersity and characterize the hydrodynamically resolved nanodisc of interest in terms of its sedimentation coefficient, Stokes’ radius and overall protein and lipid stoichiometry. Functional and biological applications are also discussed for the study of the membrane protein embedded in nanodiscs under defined experimental conditions. PMID:23219517

  20. Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis.

    PubMed

    Weber, Mary M; Bauler, Laura D; Lam, Jennifer; Hackstadt, Ted

    2015-12-01

    Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host. PMID:26416906

  1. 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

  2. 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.

  3. "SP-G", a putative new surfactant protein--tissue localization and 3D structure.

    PubMed

    Rausch, Felix; Schicht, Martin; Paulsen, Friedrich; Ngueya, Ivan; Bräuer, Lars; Brandt, Wolfgang

    2012-01-01

    Surfactant proteins (SP) are well known from human lung. These proteins assist the formation of a monolayer of surface-active phospholipids at the liquid-air interface of the alveolar lining, play a major role in lowering the surface tension of interfaces, and have functions in innate and adaptive immune defense. During recent years it became obvious that SPs are also part of other tissues and fluids such as tear fluid, gingiva, saliva, the nasolacrimal system, and kidney. Recently, a putative new surfactant protein (SFTA2 or SP-G) was identified, which has no sequence or structural identity to the already know surfactant proteins. In this work, computational chemistry and molecular-biological methods were combined to localize and characterize SP-G. With the help of a protein structure model, specific antibodies were obtained which allowed the detection of SP-G not only on mRNA but also on protein level. The localization of this protein in different human tissues, sequence based prediction tools for posttranslational modifications and molecular dynamic simulations reveal that SP-G has physicochemical properties similar to the already known surfactant proteins B and C. This includes also the possibility of interactions with lipid systems and with that, a potential surface-regulatory feature of SP-G. In conclusion, the results indicate SP-G as a new surfactant protein which represents an until now unknown surfactant protein class. PMID:23094088

  4. Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera.

    PubMed

    Kumar, Suresh

    2015-01-01

    Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO2 assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency. PMID:25780274

  5. Class III viral membrane fusion proteins

    PubMed Central

    Backovic, Marija

    2010-01-01

    SUMMARY Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane-interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry. PMID:19356922

  6. 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.

  7. Crystal structure of the TK2203 protein from Thermococcus kodakarensis, a putative extradiol dioxygenase.

    PubMed

    Nishitani, Yuichi; Simons, Jan Robert; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

    2016-06-01

    The TK2203 protein from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (262 residues, 29 kDa) is a putative extradiol dioxygenase catalyzing the cleavage of C-C bonds in catechol derivatives. It contains three metal-binding residues, but has no significant sequence similarity to proteins for which structures have been determined. Here, the first crystal structure of the TK2203 protein was determined at 1.41 Å resolution to investigate its functional role. Structure analysis reveals that this protein shares the same fold and catalytic residues as other extradiol dioxygenases, strongly suggesting the same enzymatic activity. Furthermore, the important region contributing to substrate selectivity is discussed. PMID:27303894

  8. Helix insertion into bilayers and the evolution of membrane proteins

    PubMed Central

    2010-01-01

    Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed. PMID:20039094

  9. 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.

  10. 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

  11. Membrane protein structure from rotational diffusion☆

    PubMed Central

    Das, Bibhuti B.; Park, Sang Ho; Opella, Stanley J.

    2014-01-01

    The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole–dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. PMID:24747039

  12. Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer*S⃞

    PubMed Central

    Umar, Arzu; Kang, Hyuk; Timmermans, Annemieke M.; Look, Maxime P.; Meijer-van Gelder, Marion E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W. M.; Luider, Theo M.; Foekens, John A.; Paša-Tolić, Ljiljana

    2009-01-01

    Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy

  13. 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. PMID:23291355

  14. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    PubMed

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells. PMID:26657738

  15. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  16. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane. PMID:22729905

  17. 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

  18. Identifying the hub proteins from complicated membrane protein network systems.

    PubMed

    Shen, Yi-Zhen; Ding, Yong-Sheng; Gu, Quan; Chou, Kuo-Chen

    2010-05-01

    The so-called "hub proteins" are those proteins in a protein-protein interaction network system that have remarkably higher interaction relations (or degrees) than the others. Therefore, the information of hub proteins can provide very useful insights for selecting or prioritizing targets during drug development. In this paper, by combining the multi-agent-based method with the graphical spectrum analysis and immune-genetic algorithm, a novel simulator for identifying the hub proteins from membrane protein interaction networks is proposed. As a demonstration of using the simulator, two hub membrane proteins, YPL227C and YIL147C, were identified from a complicated network system consisting of 1500 membrane proteins. Meanwhile, along with the two identified hub proteins, their molecular functions, biological processes, and cellular components were also revealed. It is anticipated that the hub-protein-simulator may become a very useful tool for system biology and drug development, particularly in deciphering unknown protein functions, determining protein complexes, and in identifying the key targets from a complicated disease system. PMID:20507268

  19. Characterization and purification of proteins which bind high-density lipoprotein. A putative cell-surface receptor.

    PubMed Central

    Bond, H M; Morrone, G; Venuta, S; Howell, K E

    1991-01-01

    High-density lipoprotein (HDL) is shown by ligand blotting to bind membrane-associated polypeptides with sizes of 60, 100 and 210 kDa. Binding was concentration-dependent and competed by excess unlabelled HDL. All the major apolipoproteins of HDL, apoA-I, apoA-II and apoA-IV, bound independently. The 100 kDa and 210 kDa HDL-binding activities were purified from membranes of Hep3B tumour cells by ion-exchange chromatography and gel filtration. The binding activities at 100 kDa and 210 kDa co-purified. After treatment with disulphide-reducing reagent, the 210 kDa band was no longer present and an increase was observed in the amount and binding ability of the 100 kDa polypeptide. The 100 kDa binding protein labelled at the cell surface with 125I could be immunoprecipitated after cross-linking to cell-surface-bound HDL. It is proposed that this HDL-binding activity, a putative cell-surface receptor for HDL, exists totally or in part as a high-molecular-mass complex composed of 100 kDa subunits. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1659384

  20. 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.

  1. 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.

  2. Transmembrane protein sorting driven by membrane curvature

    PubMed Central

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

    2015-01-01

    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. PMID:26522943

  3. Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

    PubMed

    Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

    2016-09-01

    A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1]. PMID:27583342

  4. Bio::Homology::InterologWalk - A Perl module to build putative protein-protein interaction networks through interolog mapping

    PubMed Central

    2011-01-01

    Background Protein-protein interaction (PPI) data are widely used to generate network models that aim to describe the relationships between proteins in biological systems. The fidelity and completeness of such networks is primarily limited by the paucity of protein interaction information and by the restriction of most of these data to just a few widely studied experimental organisms. In order to extend the utility of existing PPIs, computational methods can be used that exploit functional conservation between orthologous proteins across taxa to predict putative PPIs or 'interologs'. To date most interolog prediction efforts have been restricted to specific biological domains with fixed underlying data sources and there are no software tools available that provide a generalised framework for 'on-the-fly' interolog prediction. Results We introduce Bio::Homology::InterologWalk, a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. We show the application of our interolog prediction method to the genomic interactome of the fruit fly, Drosophila melanogaster. We analyse the resulting interaction networks and show that the method proposes new interactome members and interactions that are candidates for future experimental investigation. Conclusions Our interolog prediction tool employs the Ensembl Perl API and PSICQUIC enabled protein interaction data sources to generate up to date interologs 'on-the-fly'. This represents a significant advance on previous methods for interolog prediction as it allows the use of the latest orthology and protein interaction data for all of the genomes in Ensembl. The module outputs simple text files, making it easy to customise the results by

  5. Inhibition of mitochondrial membrane permeability as a putative pharmacological target for cardioprotection

    PubMed Central

    Morin, Didier; Assaly, Rana; Paradis, Stéphanie; Berdeaux, Alain

    2009-01-01

    Myocardial ischemia-reperfusion injury is a major cause of morbidity and mortality in developed countries. To date, the only treatment of complete ischemia is to restore blood flow; thus the search for new cardioprotective approaches is absolutely necessary to reduce the mortality associated with myocardial ischemia. Ischemia has long been considered to result in necrotic tissue damage but the reduction in oxygen supply can also lead to apoptosis. Therefore, in the last few years, mitochondria have become the subject of growing interest in myocardial ischemia-reperfusion since they are strongly involved in the regulation of the apoptotic process. Indeed, during ischemia-reperfusion, pathological signals converge in the mitochondria to induce permeabilization of the mitochondrial membrane. Two classes of mechanisms, which are not mutually exclusive, emerged to explain mitochondrial membrane permeabilization. The first occurs via a non-specific channel known as the mitochondrial permeability transition pore (mPTP) in the inner and the outer membranes causing disruption of the impermeability of the inner membrane, and ultimately complete inhibition of mitochondrial function. The second mechanism, involving only the outer membrane, induces the release of cell death effectors. Thus, drugs able to block or to limit mitochondrial membrane permeabilization may be cytoprotective during ischemia-reperfusion. The objective of this review is to examine the pharmacological strategies capable of inhibiting mitochondrial membrane permeabilization induced by myocardial ischemia-reperfusion. PMID:19835566

  6. Putative fasciclin-like arabinogalactan-proteins (FLA) in wheat (Triticum aestivum) and rice (Oryza sativa): identification and bioinformatic analyses.

    PubMed

    Faik, Ahmed; Abouzouhair, Jaouad; Sarhan, Fathey

    2006-11-01

    Putative plant adhesion molecules include arabinogalactan-proteins having fasciclin-like domains. In animal, fasciclin proteins participate in cell adhesion and communication. However, the molecular basis of interactions in plants is still unknown and none of these domains have been characterized in cereals. This work reports the characterization of 34 wheat (Triticum aestivum) and 24 rice (Oryza sativa) Fasciclin-Like Arabinogalactan-proteins (FLAs). Bioinformatics analyses show that cereal FLAs share structural characteristics with known Arabidopsis FLAs including arabinogalactan-protein and fasciclin conserved domains. At least 70% of the wheat and rice FLAs are predicted to be glycosylphosphatidylinositol-anchored to the plasma membranes. Expression analyses determined from the relative abundance of ESTs in the publicly available wheat EST databases and from RNA gel blots indicate that most of these genes are weakly expressed and found mainly in seeds and roots. Furthermore, most wheat genes were down regulated by abiotic stresses except for TaFLA9 and 12 where cold treatment induces their expression in roots. Plant fasciclin-like domains were predicted to have 3-D homology with FAS1 domain of the fasciclin I insect neural cell adhesion molecule with an estimated precision above 70%. The structural analysis shows that negatively charged amino acids are concentrated along the beta1-alpha3-alpha4-beta2 edges, while the positively charged amino acids are concentrated on the back side of the folds. This highly charged surface distribution could provide a way of mediating protein-protein interactions via electrostatic forces similar to many other adhesion molecules. The identification of wheat FLAs will facilitate studying their function in plant growth and development and their role in stress response. PMID:16944204

  7. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. PMID:27273628

  8. Protein permeation through an electrically tunable membrane.

    PubMed

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

    2016-05-20

    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. PMID:27044064

  9. Outer membrane proteins of Methylococcus capsulatus (Bath).

    PubMed

    Fjellbirkeland, A; Kleivdal, H; Joergensen, C; Thestrup, H; Jensen, H B

    1997-08-01

    Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl. PMID:9238104

  10. 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

  11. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum

    PubMed Central

    SUZUKI, Kenta; NISHIYAMA, Keita; MIYAJIMA, Hiroki; OSAWA, Ro; YAMAMOTO, Yuji; MUKAI, Takao

    2015-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10−8 M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  12. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

    PubMed

    Suzuki, Kenta; Nishiyama, Keita; Miyajima, Hiroki; Osawa, Ro; Yamamoto, Yuji; Mukai, Takao

    2016-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10(-8) M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  13. A Survey of Membrane Proteins in Human Serum

    PubMed Central

    Dung, Nguyen Tien; Van Chi, Phan

    2012-01-01

    Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases. PMID:25288886

  14. Proteome analysis of Paenibacillus larvae reveals the existence of a putative S-layer protein.

    PubMed

    Fünfhaus, Anne; Genersch, Elke

    2012-04-01

    Honey bee pathology has attracted much interest recently due to the problems with honey bee declines in many regions of the world. American Foulbrood (AFB) caused by Paenibacillus larvae is the most devastating bacterial brood disease of the Western honey bee (Apis mellifera) causing considerable economic losses to beekeepers worldwide. AFB outbreaks are mainly caused by two differentially virulent genotypes of P. larvae, P. larvae ERIC I and ERIC II. To better understand AFB pathogenesis and to complement already existing data from the genetic level we aimed at obtaining expression data from the protein level. We successfully developed a protocol for two-dimensional proteome analysis of P. larvae with subsequent mass-spectrometry based protein sequencing. Based on the obtained master protein maps of P. larvae genotypes ERIC I and II we identified the dominantly expressed cytosolic proteins of both genotypes, some of them presumably linked to pathogenesis and virulence. Comparing the master maps of both genotypes revealed differentially expressed proteins, i.e. a putative S-layer protein which is expressed by P. larvae ERIC II but absent from the proteome of P. larvae ERIC I. The implications of our findings for pathogenesis of AFB and virulence of P. larvae will be discussed. PMID:23757273

  15. Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

    PubMed Central

    Sanfaçon, Hélène

    2013-01-01

    Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain transmembrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase) proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C, and 3A membrane proteins. The identification of membrane association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes, and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted. PMID:23439982

  16. EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

    PubMed Central

    2011-01-01

    Background Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. Results A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal

  17. Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

    PubMed

    Hardham, J M; Stamm, L V; Porcella, S F; Frye, J G; Barnes, N Y; Howell, J K; Mueller, S L; Radolf, J D; Weinstock, G M; Norris, S J

    1997-09-15

    We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp. PMID:9332349

  18. Role of a geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity.

    PubMed

    Chowda-Reddy, R V; Achenjang, Fidelis; Felton, Christian; Etarock, Marie T; Anangfac, Marie-Therese; Nugent, Patricia; Fondong, Vincent N

    2008-07-01

    Virus-derived genes or genome fragments are increasingly being used to generate transgenic plants with resistance to plant viruses. There is need to rapidly investigate these genes in plants using transient expression prior to using them as transgenes since they may be pathogenic to plants. In this study, we investigated the AV2 protein encoded by East African cassava mosaic Cameroon virus, a virus associated with a cassava disease epidemic in western Africa. For subcellular localization, AV2 was fused to the yellow fluorescent protein (YFP) and expressed in Nicotiana benthamiana. Confocal analyses showed that AV2-YFP localizes mainly in the cytoplasm. Because it overlaps with the coat protein gene and therefore could be used to generate transgenic plants for resistance to geminiviruses, we investigated its pathogenesis in N. benthamiana by using the Potato virus X (PVX) vector. The chimeric virus PVX-AV2 induced a mild mottling in infected plants and was shown to suppress virus-induced gene silencing (VIGS). Using point mutations, we show here that AV2 pathogenicity is dependent on a conserved putative protein kinase C (PKC) phosphorylation motif. Because of its pathogenicity and ability to suppress RNA silencing, AV2 transgenic plants will less likely provide a control to geminiviruses, indeed it may weaken the resistance of the plant. We therefore suggest the use of the AV2 putative PKC mutants to generate transgenic plants. PMID:18405995

  19. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  20. 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

  1. Characterization of a putative S locus encoded receptor protein and its role in self-incompatibility

    SciTech Connect

    Not Available

    1991-01-01

    Monoclonal antibodies (MAb) were raised against purified SLSG and polyclonal antisera were raised against purified trpE/SLR1 fusion proteins. MAbH8 reacts with a protein epitope on SLSG. MAbH8 and the anit-SLR1 antisera were used with immunogold labeling to show SLSG and SLR1 proteins are localized in papillar cell walls in the stigma. MAbH8 reacts with SLSG from CRM+ cells but not CRM-cells; amino acid sequence identity between the two classes was only 65%, vs. 80% within the CRM+ class. SLSG is necessary but not sufficient for self-incompatibility. Variable molecular weight (MW) SLSG proteins are derived from the same SLG gene. MW variations in both SLSG and SLR1 are due to changes in glycosylation and phosphorylation state. SLSG is not detectable in mature pollen, but is expressed during microspore development. Using a SLG probe, a gene for a putative receptor with protein kinase activity was identified.

  2. Putative Rust Fungal Effector Proteins in Infected Bean and Soybean Leaves.

    PubMed

    Cooper, Bret; Campbell, Kimberly B; Beard, Hunter S; Garrett, Wesley M; Islam, Nazrul

    2016-05-01

    The plant-pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but their effector repertoires are not understood. The discovery of rust fungus effectors may eventually help guide decisions and actions that mitigate crop production loss. Therefore, we used mass spectrometry to identify thousands of proteins in infected beans and soybeans and in germinated fungal spores. The comparative analysis between the two helped differentiate a set of 24 U. appendiculatus proteins targeted for secretion that were specifically found in infected beans and a set of 34 U. appendiculatus proteins targeted for secretion that were found in germinated spores and infected beans. The proteins specific to infected beans included family 26 and family 76 glycoside hydrolases that may contribute to degrading plant cell walls. There were also several types of proteins with structural motifs that may aid in stabilizing the specialized fungal haustorium cell that interfaces the plant cell membrane during infection. There were 16 P. pachyrhizi proteins targeted for secretion that were found in infected soybeans, and many of these proteins resembled the U. appendiculatus proteins found in infected beans, which implies that these proteins are important to rust fungal pathology in general. This data set provides insight to the biochemical mechanisms that rust fungi use to overcome plant immune systems and to parasitize cells. PMID:26780434

  3. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins.

    PubMed

    Kralt, Annemarie; Jagalur, Noorjahan B; van den Boom, Vincent; Lokareddy, Ravi K; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M

    2015-09-15

    Endoplasmic reticulum-synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  4. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    PubMed Central

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  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. 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

  7. 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

  8. SNARE proteins and ‘membrane rafts’

    PubMed Central

    Lang, Thorsten

    2007-01-01

    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’. PMID:17478530

  9. Membrane protein structure determination by electron crystallography

    PubMed Central

    Ubarretxena-Belandia, Iban; Stokes, David L.

    2012-01-01

    During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. PMID:22572457

  10. 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.

  11. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  12. Protein-ligand and membrane-ligand interactions in pharmacology: the case of the translocator protein (TSPO).

    PubMed

    Hatty, Claire R; Banati, Richard B

    2015-10-01

    The targets of many small molecule drugs are membrane proteins, and traditionally the focus of pharmacology is on the interaction between such receptors and their small molecule drug ligands. However, the lipid membranes of cells and organelles are increasingly appreciated as diverse and dynamic structures that also specifically interact with small molecule drugs and peptides, causing profound changes in the properties of these membranes, and modulating the function of the membrane and the proteins within it. Drug-membrane interactions are likely to have a role in both the therapeutic and toxic activity of a variety of compounds, and their role in the overall pharmacological effect of a drug needs to be understood more clearly. This is the case for the 18 kDa translocator protein (TSPO) and its ligands, where functions that were established based on pharmacological studies are being called into question. Re-examining the putative functions of the TSPO and the effects of its ligands reveals a need to consider in more detail the interplay between protein-ligand and membrane-ligand interactions, and the modulatory relationship between TSPO and the lipid membrane. PMID:26238176

  13. Golgi protein FAPP2 tubulates membranes

    PubMed Central

    Cao, Xinwang; Coskun, Ünal; Rössle, Manfred; Buschhorn, Sabine B.; Grzybek, Michal; Dafforn, Timothy R.; Lenoir, Marc; Overduin, Michael; Simons, Kai

    2009-01-01

    The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network. PMID:19940249

  14. Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes

    SciTech Connect

    Wenman, W.M.; Meuser, R.U.

    1986-02-01

    Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L/sub 2/ were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. These data suggest that these putative chlamydial adhesions play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.

  15. Exploiting Microbeams for Membrane Protein Structure Determination.

    PubMed

    Warren, Anna J; Axford, Danny; Paterson, Neil G; Owen, Robin L

    2016-01-01

    A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 - http://blanco.biomol.uci.edu/mpstruc ). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography. PMID:27553238

  16. The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

    PubMed Central

    Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran

    2005-01-01

    Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027

  17. Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

    PubMed

    Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

    2015-11-01

    The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. PMID:26353761

  18. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

    PubMed

    Gong, Xiaoyan; Hurtado, Oscar; Wang, Baohua; Wu, Congqing; Yi, Mihwa; Giraldo, Martha; Valent, Barbara; Goodin, Michael; Farman, Mark

    2015-02-01

    As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane. PMID:25390188

  19. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    PubMed

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-01

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. PMID:26211889

  20. Serial Millisecond Crystallography of Membrane Proteins.

    PubMed

    Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

    2016-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage. PMID:27553240

  1. Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain.

    PubMed

    Mouille, Grégory; Ralet, Marie-Christine; Cavelier, Céline; Eland, Cathlene; Effroy, Delphine; Hématy, Kian; McCartney, Lesley; Truong, Hoai Nam; Gaudon, Virginie; Thibault, Jean-François; Marchant, Alan; Höfte, Herman

    2007-05-01

    Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions. PMID:17425712

  2. Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

    PubMed

    Sritharan, K C; Quinn, A S; Taatjes, D J; Jena, B P

    1998-01-01

    The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes. PMID:10452835

  3. Lipidic phase membrane protein serial femtosecond crystallography

    PubMed Central

    Johansson, Linda C; Arnlund, David; White, Thomas A; Katona, Gergely; DePonte, Daniel P; Weierstall, Uwe; Doak, R Bruce; Shoeman, Robert L; Lomb, Lukas; Malmerberg, Erik; Davidsson, Jan; Nass, Karol; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Bozek, John D; Caleman, Carl; Coffee, Ryan; Coppola, Nicola; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Robert; Hartmann, Andreas; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kimmel, Nils; Kirian, Richard A; Maia, Filipe R N C; Marchesini, Stefano; Martin, Andrew V; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Sierra, Raymond G; Soltau, Heike; Starodub, Dmitri; Stellato, Francesco; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wahlgren, Weixiao Y; Wang, Xiaoyu; Weidenspointner, Georg; Wunderer, Cornelia; Fromme, Petra; Chapman, Henry N; Spence, John C H; Neutze, Richard

    2012-01-01

    X-ray free electron laser (X-feL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-feL beam using a sponge phase micro-jet. PMID:22286383

  4. Theoretical analysis of protein organization in lipid membranes.

    PubMed

    Gil, T; Ipsen, J H; Mouritsen, O G; Sabra, M C; Sperotto, M M; Zuckermann, M J

    1998-11-10

    The fundamental physical principles of the lateral organization of trans-membrane proteins and peptides as well as peripheral membrane proteins and enzymes are considered from the point of view of the lipid-bilayer membrane, its structure, dynamics, and cooperative phenomena. Based on a variety of theoretical considerations and model calculations, the nature of lipid-protein interactions is considered both for a single protein and an assembly of proteins that can lead to aggregation and protein crystallization in the plane of the membrane. Phenomena discussed include lipid sorting and selectivity at protein surfaces, protein-lipid phase equilibria, lipid-mediated protein-protein interactions, wetting and capillary condensation as means of protein organization, mechanisms of two-dimensional protein crystallization, as well as non-equilibrium organization of active proteins in membranes. The theoretical findings are compared with a variety of experimental data. PMID:9804966

  5. A putative acyl-CoA-binding protein is a major phloem sap protein in rice (Oryza sativa L.).

    PubMed

    Suzui, Nobuo; Nakamura, Shin-ichi; Fujiwara, Toru; Hayashi, Hiroaki; Yoneyama, Tadakatsu

    2006-01-01

    The N-terminal amino-acid sequence of a major rice phloem-sap protein, named RPP10, was determined. RPP10 is encoded by a single gene in the rice genome. Its complete amino-acid sequence, predicted from the corresponding rice full-length cDNA, showed high similarity to plant acyl-CoA-binding proteins (ACBPs). Western blot analysis using anti-ACBP antiserum revealed that putative ACBP is abundant in the phloem sap of rice plants, and is also present in sieve-tube exudates of winter squash (Cucurbita maxima), oilseed rape (Brassica napus), and coconut palm (Cocos nucifera). These findings give rise to the idea that ACBP may involve lipid metabolism and regulation in the phloem. PMID:16804052

  6. Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

    PubMed Central

    Candau, R; Moore, P A; Wang, L; Barlev, N; Ying, C Y; Rosen, C A; Berger, S L

    1996-01-01

    Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation. PMID:8552087

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

    PubMed Central

    Simunovic, Mijo; Voth, Gregory A.

    2015-01-01

    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. PMID:26008710

  8. Protein-protein structure prediction by scoring molecular dynamics trajectories of putative poses.

    PubMed

    Sarti, Edoardo; Gladich, Ivan; Zamuner, Stefano; Correia, Bruno E; Laio, Alessandro

    2016-09-01

    The prediction of protein-protein interactions and their structural configuration remains a largely unsolved problem. Most of the algorithms aimed at finding the native conformation of a protein complex starting from the structure of its monomers are based on searching the structure corresponding to the global minimum of a suitable scoring function. However, protein complexes are often highly flexible, with mobile side chains and transient contacts due to thermal fluctuations. Flexibility can be neglected if one aims at finding quickly the approximate structure of the native complex, but may play a role in structure refinement, and in discriminating solutions characterized by similar scores. We here benchmark the capability of some state-of-the-art scoring functions (BACH-SixthSense, PIE/PISA and Rosetta) in discriminating finite-temperature ensembles of structures corresponding to the native state and to non-native configurations. We produce the ensembles by running thousands of molecular dynamics simulations in explicit solvent starting from poses generated by rigid docking and optimized in vacuum. We find that while Rosetta outperformed the other two scoring functions in scoring the structures in vacuum, BACH-SixthSense and PIE/PISA perform better in distinguishing near-native ensembles of structures generated by molecular dynamics in explicit solvent. Proteins 2016; 84:1312-1320. © 2016 Wiley Periodicals, Inc. PMID:27253756

  9. 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

  10. A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens.

    PubMed

    Mehta, Teena; Childers, Susan E; Glaven, Richard; Lovley, Derek R; Mester, Tünde

    2006-08-01

    Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species. PMID:16849792

  11. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

  12. Nramp defines a family of membrane proteins.

    PubMed Central

    Cellier, M; Privé, G; Belouchi, A; Kwan, T; Rodrigues, V; Chia, W; Gros, P

    1995-01-01

    Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants previously described in ion channels. These characteristics suggest that the Nramp polypeptides form part of a group of transporters or channels that act on as yet unidentified substrates. Images Fig. 1 PMID:7479731

  13. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

    PubMed Central

    Londono-Renteria, Berlin; Troupin, Andrea; Conway, Michael J; Vesely, Diana; Ledizet, Michael; Roundy, Christopher M.; Cloherty, Erin; Jameson, Samuel; Vanlandingham, Dana; Higgs, Stephen; Fikrig, Erol; Colpitts, Tonya M.

    2015-01-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses. PMID:26491875

  14. Amphipathic agents for membrane protein study.

    PubMed

    Sadaf, Aiman; Cho, Kyung Ho; Byrne, Bernadette; Chae, Pil Seok

    2015-01-01

    Membrane proteins (MPs) are insoluble in aqueous media as a result of incompatibility between the hydrophilic property of the solvent molecules and the hydrophobic nature of MP surfaces, normally associated with lipid membranes. Amphipathic compounds are necessary for extraction of these macromolecules from the native membranes and their maintenance in solution. The amphipathic agents surround the hydrophobic segments of MPs, thus serving as a membrane mimetic system. Of the available amphipathic agents, detergents are most widely used for MP manipulation. However, MPs encapsulated by conventional detergent micelles have a tendency to undergo structural degradation, hampering MP advance, and necessitating the development of novel detergents with enhanced efficacy for MP study. In this chapter, we will introduce both conventional and novel classes of detergents and discuss about the chemical structures, design principles, and efficacies of these compounds for MP solubilization and stabilization. The behaviors of those agents toward MP crystallization will be a primary topic in our discussion. This discussion highlights the common features of popular conventional/novel detergents essential for successful MP structural study. The conclusions reached by this discussion would not only enable MP scientists to rationally select a set of detergent candidates among a large number of detergents but also provide detergent inventors with useful guidelines in designing novel amphipathic systems. PMID:25950960

  15. 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. PMID:27547605

  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. 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

  18. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    NASA Astrophysics Data System (ADS)

    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.

  19. Membrane protein assembly: genetic, evolutionary and medical perspectives.

    PubMed

    Manoil, C; Traxler, B

    1995-01-01

    Lipid bilayers are delicate structures that are easily disrupted by a variety of amphipathic molecules. Yet the viability of a cell requires the continued assembly of large amphipathic proteins within its membranes without damage. The need to minimize bilayer disruption may account for a number of fundamental features of membrane protein assembly. These include the use of redundant sequence information to establish the topologies and folded structures of membrane proteins, and the existence of efficient mechanisms to rid cells of misassembled proteins. Most missense mutations that inactivate a membrane protein probably do so by altering the folding of the membrane-inserted structure rather than by rearranging the topology or by changing key residues involved directly in function. Such misfolded membrane proteins may be toxic to cells if they escape cellular safeguards. This toxicity may underlie some human degenerative diseases due to mutant membrane proteins. PMID:8825471

  20. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  1. A saposin-lipoprotein nanoparticle system for membrane proteins.

    PubMed

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

    2016-04-01

    A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state. PMID:26950744

  2. 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.

  3. 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.

  4. Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus

    PubMed Central

    Vives-Adrián, Laia; Garriga, Damià; Buxaderas, Mònica; Fraga, Joana; Pereira, Pedro José Barbosa

    2015-01-01

    ABSTRACT The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family. PMID:25589659

  5. Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

    PubMed

    Cheng, Chi-Yuan; Han, Songi

    2013-01-01

    Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments. PMID:23331309

  6. Dynamic Nuclear Polarization Methods in Solids and Solutions to Explore Membrane Proteins and Membrane Systems

    NASA Astrophysics Data System (ADS)

    Cheng, Chi-Yuan; Han, Songi

    2013-04-01

    Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

  7. 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

  8. 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

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

    PubMed

    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

  10. 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

  11. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    PubMed Central

    2012-01-01

    Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

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

    PubMed Central

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

    2015-01-01

    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. PMID:25514890

  13. 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

  14. Leafy head2, which encodes a putative RNA-binding protein, regulates shoot development of rice.

    PubMed

    Xiong, Guo Sheng; Hu, Xing Ming; Jiao, Yong Qing; Yu, Yan Chun; Chu, Cheng Cai; Li, Jia Yang; Qian, Qian; Wang, Yong Hong

    2006-03-01

    During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 (lhd2) mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TE1. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass. PMID:16541125

  15. Crystal structure of a putative oligopeptide-binding periplasmic protein from a hyperthermophile.

    PubMed

    Yoon, Hye-Jin; Kim, Hee Jung; Mikami, Bunzo; Yu, Yeon Gyu; Lee, Hyung Ho

    2016-09-01

    Oligopeptide-binding proteins (Opps) are part of the ATP-binding cassette system, playing a crucial role in nutrient uptake and sensing the external environment in bacteria, including hyperthermophiles. Opps serve as a binding platform for diverse peptides; however, how these peptides are recognized by Opps is still largely unknown and few crystal structures of Opps from hyperthermophiles have been determined. To facilitate such an understanding, the crystal structure of a putative Opp, OppA from Thermotoga maritima (TmOppA), was solved at 2.6-Å resolution in the open conformation. TmOppA is composed of three domains. The N-terminal domain consists of twelve strands, nine helices, and four 310 helices, and the C-terminal domain consists of five strands, ten helices, and one 310 helix. These two domains are connected by the linker domain, which consists of two strands, three helices, and three 310 helices. Based on structural comparisons of TmOppA with other OppAs and binding studies, we suggest that TmOppA might be a periplasmic Opp. The most distinct feature of TmOppA is the insertion of two helices, which are lacking in other OppAs. A cavity volume between the N-terminal and C-terminal domains is suggested to be responsible for binding peptides of various lengths. PMID:27377296

  16. Protein crystals on phase-separating model membranes

    NASA Astrophysics Data System (ADS)

    Manley, Suliana; Horton, Margaret; Leszczynski, Szymon; Gast, Alice

    2006-03-01

    We study the interplay between the crystallization of proteins tethered to membranes and separation within the membranes of giant unilamellar vesicles (GUVs) composed of DOPC, sphingomyelin (SM), and cholesterol. These model membranes phase separate into coexisting liquid domains below a miscibility transition temperature. This phase separation captures some aspects of the formation of lipid rafts in cell membranes and demonstrates the influence of membrane composition on raft formation. Real cell membranes have a much more complicated structure. There are additional physical constraints present in cell membranes, such as their attachment to the cytoskeleton and the presence of membrane bound proteins. The self-association of membrane proteins can influence the membrane phase behavior. We begin to investigate these effects on model tethered protein- loaded membranes by incorporating a small amount of biotin-X- DPPE into our GUVs. The biotinylated lipid partitions into a cholesterol-poor phase; thus, streptavidin binds preferentially to one of the membrane phases. As streptavidin assembles to form crystalline domains, it restricts the membrane mobility. We examine the effect of this protein association on lipid phase separation, as well as the effect of the lipid phase separation on the crystallization of the tethered proteins.

  17. Membrane Protein Structure and Dynamics from NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Hong, Mei; Zhang, Yuan; Hu, Fanghao

    2012-05-01

    We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca2+ pumps, G protein-coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.

  18. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2013-06-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein-coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy.

  19. 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

  20. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  1. 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

  2. Diffusing proteins on a fluctuating membrane: Analytical theory and simulations

    NASA Astrophysics Data System (ADS)

    Reister-Gottfried, Ellen; Leitenberger, Stefan M.; Seifert, Udo

    2010-03-01

    Using analytical calculations and computer simulations, we consider both the lateral diffusion of a membrane protein and the fluctuation spectrum of the membrane in which the protein is embedded. The membrane protein interacts with the membrane shape through its spontaneous curvature and bending rigidity. The lateral motion of the protein may be viewed as diffusion in an effective potential, hence, the effective mobility is always reduced compared to the case of free diffusion. Using a rigorous path-integral approach, we derive an analytical expression for the effective diffusion coefficient for small ratios of temperature and bending rigidity, which is the biologically relevant limit. Simulations show very good quantitative agreement with our analytical result. The analysis of the correlation functions contributing to the diffusion coefficient shows that the correlations between the stochastic force of the protein and the response in the membrane shape are responsible for the reduction. Our quantitative analysis of the membrane height correlation spectrum shows an influence of the protein-membrane interaction causing a distinctly altered wave-vector dependence compared to a free membrane. Furthermore, the time correlations exhibit the two relevant time scales of the system: that of membrane fluctuations and that of lateral protein diffusion with the latter typically much longer than the former. We argue that the analysis of the long-time decay of membrane height correlations can thus provide a new means to determine the effective diffusion coefficient of proteins in the membrane.

  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. Graphical Abstract The legend of online abstract figure is (a) schematic illustration of membrane proteins extraction, purification and digestion from living cells; (b) diagrammatic sketch of the automatic integrated membrane proteome analysis system. PMID:26922343

  4. Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

    PubMed

    Kuznetsov, Andrey S; Polyansky, Anton A; Fleck, Markus; Volynsky, Pavel E; Efremov, Roman G

    2015-09-01

    The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association. PMID:26575933

  5. Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

    PubMed Central

    Besingi, Richard N.; Clark, Patricia L.

    2016-01-01

    Membrane proteins play crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but is rapidly digested when cells are lysed prior to evaluation. Reporter proteins that are resistant to proteases (e.g. maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g. SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h. PMID:26584447

  6. 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.

  7. 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. PMID:25828256

  8. Holophytochrome-Interacting Proteins in Physcomitrella: Putative Actors in Phytochrome Cytoplasmic Signaling

    PubMed Central

    Ermert, Anna Lena; Mailliet, Katharina; Hughes, Jon

    2016-01-01

    Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP’s) and their possible roles in signaling. PMID:27242820

  9. Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

    PubMed

    Bogdanov, Mikhail; Heacock, Philip N; Dowhan, William

    2010-01-01

    A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system. PMID:20419405

  10. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Opella, Stanley J.

    2014-01-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein–coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy. PMID:23577669

  11. Membrane-Mediated Interaction between Strongly Anisotropic Protein Scaffolds

    PubMed Central

    Schweitzer, Yonatan; Kozlov, Michael M.

    2015-01-01

    Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains. PMID:25710602

  12. 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

  13. Functional characterization of Pseudomonas fluorescens OprE and OprQ membrane proteins.

    PubMed

    Jaouen, Thomas; Coquet, Laurent; Marvin-Guy, Laure; Orange, Nicole; Chevalier, Sylvie; Dé, Emmanuelle

    2006-08-01

    Outer membrane (OM) proteins of the OprD family may enable bacteria of the genus Pseudomonas to adapt to various environments by modulating OM permeability. The OprE and OprQ porins from P. fluorescens strain MF0 were purified and identified by MALDI-TOF mass spectrometry and N-terminal and internal microsequencing. These proteins, when reconstituted in an artificial planar lipid bilayer, induced similar ion channels with low single-conductance values. Secondary structure prediction of both proteins showed similar folding patterns into a 16 transmembrane beta-strands barrel but a highly variable amino-acid composition and length for their putative external loops implicated in porin function. Both proteins were overexpressed under poor oxygenation conditions, but not by using several amino acids as sole carbon source, indicating a different specificity for these proteins compared to the paradigm of this protein family, OprD. PMID:16777062

  14. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana.

    PubMed

    Josefsson, L G; Rask, L

    1997-10-15

    We have cloned and characterized a cDNA from Arabidopsis thaliana that most likely encodes a novel member of the vast superfamily of G-protein-coupled receptor proteins (GPCRs). By taking advantage of amino acid sequence similarities between plant expressed sequence tags (ESTs) and established G-protein-coupled receptor sequences, a probe was obtained which was used for the screening of an Arabidopsis cDNA library. The cDNA which was found is very infrequently represented in the cDNA library, suggesting a low and/or spatially restricted expression. A region of the translated sequence of the cDNA shows the highest similarity to cAMP receptors from the slime mold Dictyostelium discoideum. The same region is also similar to that in members of the animal calcitonin family of receptors. Another region of the putative receptor, however, is similar to sequences of serotonin receptors and other receptors of the so-called rhodopsin family of GPCRs. The rhodopsin family has numerous members in higher vertebrate species. Alignments and phylogenetic analyses of the regions of similarity yielded results in accordance with other evolutionary considerations. Our cDNA thus occurred on a distinct major branch in relation to the rest of the rhodopsin family. In relation to the calcitonin family, our cDNA and cAMP receptors occurred together on a distinct major branch but appear to have diverged from each other shortly after their divergence from the rest of the calcitonin family. Other features further argue for a tentative identification of it as a GPCR. It displays seven discrete and strongly predicted transmembrane domains when analyzed in hydropathy plots. The preferred orientation is with the amino terminus towards the outside. It has one Cys residue in extracellular loop 1 and another in extracellular loop 2. Cys residues in these loops are known to form disulfide bridges in many other GPCRs. Finally, it has several fully conserved amino acids that belong to the most conserved

  15. Protein-Induced Modulation of Chloroplast Membrane Morphology

    PubMed Central

    Machettira, Anu B.; Groß, Lucia E.; Tillmann, Bodo; Weis, Benjamin L.; Englich, Gisela; Sommer, Maik S.; Königer, Martina; Schleiff, Enrico

    2012-01-01

    Organelles are surrounded by membranes with a distinct lipid and protein composition. While it is well established that lipids affect protein functioning and vice versa, it has been only recently suggested that elevated membrane protein concentrations may affect the shape and organization of membranes. We therefore analyzed the effects of high chloroplast envelope protein concentrations on membrane structures using an in vivo approach with protoplasts. Transient expression of outer envelope proteins or protein domains such as CHUP1-TM–GFP, outer envelope protein of 7 kDa–GFP, or outer envelope protein of 24 kDa–GFP at high levels led to the formation of punctate, circular, and tubular membrane protrusions. Expression of inner membrane proteins such as translocase of inner chloroplast membrane 20, isoform II (Tic20-II)–GFP led to membrane protrusions including invaginations. Using increasing amounts of DNA for transfection, we could show that the frequency, size, and intensity of these protrusions increased with protein concentration. The membrane deformations were absent after cycloheximide treatment. Co-expression of CHUP1-TM–Cherry and Tic20-II–GFP led to membrane protrusions of various shapes and sizes including some stromule-like structures, for which several functions have been proposed. Interestingly, some structures seemed to contain both proteins, while others seem to contain one protein exclusively, indicating that outer and inner envelope dynamics might be regulated independently. While it was more difficult to investigate the effects of high expression levels of membrane proteins on mitochondrial membrane shapes using confocal imaging, it was striking that the expression of the outer membrane protein Tom20 led to more elongate mitochondria. We discuss that the effect of protein concentrations on membrane structure is possibly caused by an imbalance in the lipid to protein ratio and may be involved in a signaling pathway regulating membrane

  16. The putative Notch ligand HyJagged is a transmembrane protein present in all cell types of adult Hydra and upregulated at the boundary between bud and parent

    PubMed Central

    2011-01-01

    Background The Notch signalling pathway is conserved in pre-bilaterian animals. In the Cnidarian Hydra it is involved in interstitial stem cell differentiation and in boundary formation during budding. Experimental evidence suggests that in Hydra Notch is activated by presenilin through proteolytic cleavage at the S3 site as in all animals. However, the endogenous ligand for HvNotch has not been described yet. Results We have cloned a cDNA from Hydra, which encodes a bona-fide Notch ligand with a conserved domain structure similar to that of Jagged-like Notch ligands from other animals. Hyjagged mRNA is undetectable in adult Hydra by in situ hybridisation but is strongly upregulated and easily visible at the border between bud and parent shortly before bud detachment. In contrast, HyJagged protein is found in all cell types of an adult hydra, where it localises to membranes and endosomes. Co-localisation experiments showed that it is present in the same cells as HvNotch, however not always in the same membrane structures. Conclusions The putative Notch ligand HyJagged is conserved in Cnidarians. Together with HvNotch it may be involved in the formation of the parent-bud boundary in Hydra. Moreover, protein distribution of both, HvNotch receptor and HyJagged indicate a more widespread function for these two transmembrane proteins in the adult hydra, which may be regulated by additional factors, possibly involving endocytic pathways. PMID:21899759

  17. Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes

    PubMed Central

    Wolff, Bernard J.; Morrison, Shatavia S.; Pesti, Denise; Ganakammal, Satishkumar Ranganathan; Srinivasamoorthy, Ganesh; Changayil, Shankar; Weil, M. Ryan; MacCannell, Duncan; Rowe, Lori; Frace, Michael; Ritchie, Branson W.; Dean, Deborah

    2015-01-01

    Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer-membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer-membrane proteins (Pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the Pmps and tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease. PMID:25887617

  18. 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

  19. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    PubMed

    Alford, Rebecca F; Koehler Leman, Julia; Weitzner, Brian D; Duran, Amanda M; Tilley, Drew C; Elazar, Assaf; Gray, Jeffrey J

    2015-09-01

    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

  20. Sampling the membrane: function of rhomboid-family proteins.

    PubMed

    Lemberg, Marius K

    2013-05-01

    Rhomboids constitute a conserved protein superfamily that specifically binds membrane proteins and directs them into various different cellular pathways ranging from regulated secretion to endoplasmic reticulum (ER)-associated degradation (ERAD). Rhomboid proteases are known to release protein domains from membranes by a cut in their membrane anchor, whereas an emerging new class of rhomboid-family proteins lacks key catalytic residues and is not proteolytically active. Recent work has shown that these rhomboid pseudoproteases, including iRhoms and derlins, bind membrane proteins to regulate their fate, but the underlying molecular mechanism is not known. This review summarizes recent advances in the molecular understanding of rhomboid-family proteins and discusses common principles in how they recognize and bind proteins in the plane of the membrane. PMID:23369641

  1. A topological and conformational stability alphabet for multipass membrane proteins.

    PubMed

    Feng, Xiang; Barth, Patrick

    2016-03-01

    Multipass membrane proteins perform critical signal transduction and transport across membranes. How transmembrane helix (TMH) sequences encode the topology and conformational flexibility regulating these functions remains poorly understood. Here we describe a comprehensive analysis of the sequence-structure relationships at multiple interacting TMHs from all membrane proteins with structures in the Protein Data Bank (PDB). We found that membrane proteins can be deconstructed in interacting TMH trimer units, which mostly fold into six distinct structural classes of topologies and conformations. Each class is enriched in recurrent sequence motifs from functionally unrelated proteins, revealing unforeseen consensus and evolutionary conserved networks of stabilizing interhelical contacts. Interacting TMHs' topology and local protein conformational flexibility were remarkably well predicted in a blinded fashion from the identified binding-hotspot motifs. Our results reveal universal sequence-structure principles governing the complex anatomy and plasticity of multipass membrane proteins that may guide de novo structure prediction, design, and studies of folding and dynamics. PMID:26780406

  2. Intrinsic membrane association of Drosophila cysteine string proteins.

    PubMed

    Mastrogiacomo, A; Kohan, S A; Whitelegge, J P; Gundersen, C B

    1998-09-25

    Cysteine string proteins (csps) are highly conserved constituents of vertebrate and invertebrate secretory organelles. Biochemical and immunoprecipitation experiments implied that vertebrate csps were integral membrane proteins that were tethered to the outer leaflet of secretory vesicles via the fatty acyl residues of their extensively acylated cysteine string. Independently, work of others suggested that Drosophila csps were peripheral membrane proteins that were anchored to membranes by a mechanism that was independent of the cysteine string and its fatty acyl residues. We extended these investigation and found first that sodium carbonate treatment partially stripped both csps and the integral membrane protein, synaptotagmin, from Drosophila membranes. Concomitantly, carbonate released fatty acids into the medium, arguing that it has a mild, solubilizing effect on these membranes. Second, we observed that Drosophila csps behaved like integral membrane proteins in Triton X-114 partitioning experiments. Third, we found that when membrane-bound csps were deacylated, they remained membrane bound. Moreover, it appeared that hydrophobic interactions were necessary for this persistent membrane association of csps. Thus, neither reducing conditions, urea, nor chaotropic agents displaced deacylated csps from membranes. Only detergents were effective in solubilizing deacylated csps. Finally, by virtue of the inaccessibility of deacylated csps to thiol alkylation by the membrane-impermeant alkylating reagent, iodoacetic acid, we inferred that it was the cysteine string domain that mediated the membrane association of deacylated csps. Thus, we conclude that under physiological conditions csps are integral membrane proteins of secretory organelles, and that the cysteine string domain plays a vital role in the membrane association of these proteins. PMID:9771899

  3. Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

    PubMed Central

    Hu, Wensi S.; Ng, Wailap Victor; Lu, Chi-Yu; Huang, Tsung-Yen; Shu, Hung-Wei; Hsiao, Kwang-Jen; Tsai, Shih-Feng; Chang, Chuan-Hsiung; Lin, Chao-Hsiung

    2012-01-01

    Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen. PMID:22536369

  4. Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

    PubMed

    Watson, Eleanor; Alberdi, M Pilar; Inglis, Neil F; Lainson, Alex; Porter, Megan E; Manson, Erin; Imrie, Lisa; Mclean, Kevin; Smith, David G E

    2014-12-01

    Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control. PMID:25457368

  5. The Use of Detergents to Purify Membrane Proteins.

    PubMed

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

    2016-01-01

    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. PMID:27038269

  6. 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

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

    NASA Astrophysics Data System (ADS)

    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

  8. 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

  9. 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.

  10. Towards Co-Evolution of Membrane Proteins and Metabolism

    NASA Astrophysics Data System (ADS)

    Wilson, Michael A.; Wei, Chenyu; Pohorille, Andrew

    2014-12-01

    Primordial metabolism co-evolved with the earliest membrane peptides to produce more environmentally fit progeny. Here, we map a continuous, evolutionary path that connects nascent biochemistry with simple, membrane-bound oligopeptides, ion channels and, further, membrane proteins capable of energy transduction and utilization of energy for active transport.

  11. Guided reconstitution of membrane protein fragments.

    PubMed

    Cohen, Leah S; Arshava, Boris; Kauffman, Sarah; Mathew, Elizabeth; Fracchiolla, Katrina E; Ding, Fa-Xiang; Dumont, Mark E; Becker, Jeffrey M; Naider, Fred

    2014-01-01

    Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination. PMID:23897574

  12. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    DOE PAGESBeta

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-04-28

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all

  13. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling.

    PubMed

    Barbaglia, Allison M; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  14. Phloem Proteomics Reveals New Lipid-Binding Proteins with a Putative Role in Lipid-Mediated Signaling

    PubMed Central

    Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; Hoffmann-Benning, Susanne

    2016-01-01

    Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three

  15. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  16. Anomalous diffusion of proteins in sheared lipid membranes.

    PubMed

    Khoshnood, Atefeh; Jalali, Mir Abbas

    2013-09-01

    We use coarse grained molecular dynamics simulations to investigate diffusion properties of sheared lipid membranes with embedded transmembrane proteins. In membranes without proteins, we find normal in-plane diffusion of lipids in all flow conditions. Protein embedded membranes behave quite differently: by imposing a simple shear flow and sliding the monolayers of the membrane over each other, the motion of protein clusters becomes strongly superdiffusive in the shear direction. In such a circumstance, the subdiffusion regime is predominant perpendicular to the flow. We show that superdiffusion is a result of accelerated chaotic motions of protein-lipid complexes within the membrane voids, which are generated by hydrophobic mismatch or the transport of lipids by proteins. PMID:24125292

  17. 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.

  18. 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

  19. Energy-coupled outer membrane transport proteins and regulatory proteins.

    PubMed

    Braun, Volkmar; Endriss, Franziska

    2007-06-01

    FhuA and FecA are two examples of energy-coupled outer membrane import proteins of gram-negative bacteria. FhuA transports iron complexed by the siderophore ferrichrome and serves as a receptor for phages, a toxic bacterial peptide, and a toxic protein. FecA transports diferric dicitrate and regulates transcription of an operon encoding five ferric citrate (Fec) transport genes. Properties of FhuA mutants selected according to the FhuA crystal structure are described. FhuA mutants in the TonB box, the hatch, and the beta-barrel are rather robust. TonB box mutants in FhuA FecA, FepA, Cir, and BtuB are compared; some mutations are suppressed by mutations in TonB. Mutant studies have not revealed a ferrichrome diffusion pathway, and tolerance to mutations in the region linking the TonB box to the hatch does not disclose a mechanism for how energy transfer from the cytoplasmic membrane to FhuA changes the conformation of FhuA such that bound substrates are released, the pore is opened, and substrates enter the periplasm, or how surface loops change their conformation such that TonB-dependent phages bind irreversibly and release their DNA into the cells. The FhuA and FecA crystal structures do not disclose the mechanism of these proteins, but they provide important information for specific functional studies. FecA is also a regulatory protein that transduces a signal from the cell surface into the cytoplasm. The interacting subdomains of the proteins in the FecA --> FecR --> FecI --> RNA polymerase signal transduction pathway resulting in fecABCDE transcription have been determined. Energy-coupled transporters transport not only iron and vitamin B12, but also other substrates of very low abundance such as sugars across the outer membrane; transcription regulation of the transport genes may occur similarly to that of the Fec transport genes. PMID:17370038

  20. In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite.

    PubMed

    Veenstra, Jan A; Rombauts, Stephane; Grbić, Miodrag

    2012-04-01

    The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown. PMID:22214827

  1. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  2. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  3. Single molecule techniques for the study of membrane proteins.

    PubMed

    García-Sáez, Ana J; Schwille, Petra

    2007-08-01

    Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field. PMID:17497147

  4. Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus.

    PubMed

    Li, Qing; Tang, Zhong; Hu, Yibing; Yu, Ling; Liu, Zhaopu; Xu, Guohua

    2014-08-01

    Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na(+)/H(+) antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4% identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na(+) and accumulate more K(+). Expression of HtSOS1 decreased Na(+) content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte. PMID:24771143

  5. The chloroplast membrane associated ceQORH putative quinone oxidoreductase reduces long-chain, stress-related oxidized lipids.

    PubMed

    Curien, Gilles; Giustini, Cécile; Montillet, Jean-Luc; Mas-Y-Mas, Sarah; Cobessi, David; Ferrer, Jean-Luc; Matringe, Michel; Grechkin, Alexander; Rolland, Norbert

    2016-02-01

    Under oxidative stress conditions the lipid constituents of cells can undergo oxidation whose frequent consequence is the production of highly reactive α,β-unsaturated carbonyls. These molecules are toxic because they can add to biomolecules (such as proteins and nucleic acids) and several enzyme activities cooperate to eliminate these reactive electrophile species. CeQORH (chloroplast envelope Quinone Oxidoreductase Homolog, At4g13010) is associated with the inner membrane of the chloroplast envelope and imported into the organelle by an alternative import pathway. In the present study, we show that the recombinant ceQORH exhibits the activity of a NADPH-dependent α,β-unsaturated oxoene reductase reducing the double bond of medium-chain (C⩾9) to long-chain (18 carbon atoms) reactive electrophile species deriving from poly-unsaturated fatty acid peroxides. The best substrates of ceQORH are 13-lipoxygenase-derived γ-ketols. γ-Ketols are spontaneously produced in the chloroplast from the unstable allene oxide formed in the biochemical pathway leading to 12-oxo-phytodienoic acid, a precursor of the defense hormone jasmonate. In chloroplasts, ceQORH could detoxify 13-lipoxygenase-derived γ-ketols at their production sites in the membranes. This finding opens new routes toward the understanding of γ-ketols role and detoxification. PMID:26678323

  6. Immunoassay of urinary retinol binding protein as a putative renal marker in cats.

    PubMed

    van Hoek, Ingrid; Daminet, Sylvie; Notebaert, Sofie; Janssens, Isabel; Meyer, Evelyne

    2008-01-01

    The presence of low molecular weight retinol binding protein (RBP) in urine reflects tubular damage. Therefore, RBP has been used as a renal marker in humans and dogs. Using an anti-human RBP antibody (Ab), this study first demonstrates feline urinary RBP by Western blot analysis and then evaluates its potential as a renal marker in cats by enzyme-linked immunosorbent assay (ELISA). Urine was taken by cystocentesis, centrifuged and stored at -80 degrees C until analysis. Urinary RBP levels were compared in clinically healthy cats (H), chronic renal failure patients (CRF) and cats with hyperthyroidism (HT). The detection of a band at the same position as the human RBP standard with Western blot analysis, indicated that RBP was present in the urine of CRF and HT patients but minimally present in H cats. The data obtained with ELISA were in accordance with these observations. RBP levels were expressed as RBP:creatinine (RBP:c) ratios following normalisation with urinary creatinine. The functional assay sensitivity was 1.37 microg/l RBP. Parallelism between the trend lines of the human RBP standard curve and the curves obtained from sequentially diluted urine samples indicated that feline RBP was recovered. The mean intra-assay coefficient of variance was 7% and the standardised agreement index revealed satisfactory day-to-day repeatability. The RBP:c ratio in all H cats (n=10) was below the assay sensitivity. The groups of CRF and HT patients had increased mean RBP:c ratios of 1.6+/-0.5x10(-2) microg/mg (mean+/-SEM, n=10) and 1.4+/-0.4x10(-2) microg/mg (n=13), respectively. Both groups showed a large variation in the relative RBP concentrations of individual cats. In conclusion, RBP is demonstrated for the first time in urine from most CRF and HT patients and the validated ELISA allows its evaluation as a putative renal marker in cats. PMID:17996888

  7. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  8. Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction.

    PubMed

    Pabuwal, Vagmita; Li, Zhijun

    2008-01-01

    De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction. PMID:18178566

  9. The Hydrophobic Insertion Mechanism of Membrane Curvature Generation by Proteins

    PubMed Central

    Campelo, Felix; McMahon, Harvey T.; Kozlov, Michael M.

    2008-01-01

    A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices. PMID:18515373

  10. 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.

  11. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

    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. Graphical Abstract ᅟ. PMID:27106602

  12. 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.

    2016-04-01

    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.

  13. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

    PubMed

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  14. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

    PubMed Central

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

  15. Composition fluctuations, correlated response, and protein solvation in membranes

    NASA Astrophysics Data System (ADS)

    McConnell, Harden

    2010-05-01

    Membrane composition fluctuations are deduced from the deuterium NMR relaxation data of S. L. Veatch et al. [Proc. Natl. Acad. Sci. U.S.A. 104, 17650 (2007)]. A theoretical model for these fluctuations is used to determine the parameters of a correlation function. A fluctuation-response relation is then derived to infer the response of a lipid bilayer membrane to perturbations, such as the presence of a protein. The energy of the correlated response is shown to decrease as a bilayer miscibility critical point is approached from higher temperatures. Near the critical temperature the low energy of the composition response facilitates the lipid solvation of membrane proteins and minimizes lipid-mediated nonspecific protein-protein interactions. This facilitated lipid solvation of membrane proteins may be the basis of reports that at the growth temperature, the lipids of animal cell membranes have compositions such that they are within ˜10° of a miscibility critical point.

  16. Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties▿

    PubMed Central

    Mulay, Vishwaroop; Caimano, Melissa J.; Liveris, Dionysios; Desrosiers, Daniel C.; Radolf, Justin D.; Schwartz, Ira

    2007-01-01

    The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins. PMID:17189354

  17. Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties.

    PubMed

    Mulay, Vishwaroop; Caimano, Melissa J; Liveris, Dionysios; Desrosiers, Daniel C; Radolf, Justin D; Schwartz, Ira

    2007-03-01

    The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins. PMID:17189354

  18. SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein.

    PubMed

    Jialin, Gao; Xuefan, Gu; Huiwen, Zhang

    2010-11-26

    In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression. PMID:20965152

  19. X-ray Diffraction from Membrane Protein Nanocrystals

    PubMed Central

    Hunter, M.S.; DePonte, D.P.; Shapiro, D.A.; Kirian, R.A.; Wang, X.; Starodub, D.; Marchesini, S.; Weierstall, U.; Doak, R.B.; Spence, J.C.H.; Fromme, P.

    2011-01-01

    Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells. PMID:21190672

  20. Protein quality control at the inner nuclear membrane

    PubMed Central

    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

    2015-01-01

    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 expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-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 Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest 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 (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

  1. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  2. A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba

    PubMed Central

    Santos, Herbert J.; Imai, Kenichiro; Makiuchi, Takashi; Tomii, Kentaro; Horton, Paul; Nozawa, Akira; Ibrahim, Mohamed; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-01-01

    Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes. PMID:25711150

  3. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    PubMed Central

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Müller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Günther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins. PMID:22918945

  4. Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

    PubMed

    Hallée, Stéphanie; Richard, Dave

    2015-01-01

    Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion. PMID:26375591

  5. The "putative" leucine zipper region of murine leukemia virus transmembrane protein (P15e) is essential for viral infectivity.

    PubMed

    Ramsdale, E E; Kingsman, S M; Kingsman, A J

    1996-06-01

    In order to determine the role of the putative leucine zipper region of murine leukemia virus (MLV) transmembrane protein p15E, nine mutations in this region were introduced by site-directed mutagenesis. None of these mutations affected the expression or transport of the envelope protein or incorporation into virions. The mutants were analyzed for their ability to infect NIH3T3 cells and to induce cell fusion in a rat XC cell fusion assay. Mutations removing the charge of the hydrophilic residues reduced infectivity in NIH3T3 cells but had either no effect or a minor effect on envelope-induced XC cell fusion. Six mutations of hydrophobic residues of the putative leucine zipper region were constructed; four completely abolished the ability to infect NIH3T3 cells and these mutant envelopes were also unable to induce cell fusion in the XC cell fusion assay. These data demonstrate the absolute requirement for the putative leucine zipper region for both fusion and infection of MLV. PMID:8659102

  6. 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.

  7. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  8. Gravistimulation changes expression of genes encoding putative carrier proteins of auxin polar transport in etiolated pea epicotyls

    NASA Astrophysics Data System (ADS)

    Hoshino, T.; Hitotsubashi, R.; Miyamoto, K.; Tanimoto, E.; Ueda, J.

    STS-95 space experiment has showed that auxin polar transport in etiolated epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings is controlled by gravistimulation. In Arabidopsis thaliana auxin polar transport has considered to be regulated by efflux and influx carrier proteins in plasma membranes, AtPIN1 and AtAUX1, respectively. In order to know how gravistimuli control auxin polar transport in etiolated pea epicotyls at molecular levels, strenuous efforts have been made, resulting in successful isolation of full-length cDNAs of a putative auxin efflux and influx carriers, PsPIN2 and PsAUX1, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857, Chawla and DeMason, 2003) and AtPINs, and also among PsAUX1, AtAUX1 and their related genes. Phylogenetic analyses based on the deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 relating to lateral transport of auxin, while PsPIN1 belonged to the same clade as AtPIN1 relating to auxin polar transport. In the present study, we examined the effects of gravistimuli on the expression of PsPINs and PsAUX1 in etiolated pea seedlings by northern blot analysis. Expression of PsPIN1, PsPIN2 and PsAUX1 in hook region of 3.5-d-old etiolated pea seedlings grown under simulated microgravity conditions on a 3-D clinostat increased as compared with that of the seedlings grown under 1 g conditions. On the other hand, that of PsPIN1 and PsAUX1 in the 1st internode region under simulated microgravity conditions on a 3-D clinostat also increased, while that of PsPIN2 was affected little. These results suggest that expression of PsPIN1, PsPIN2 and PsAUX1 regulating polar/lateral transport of auxin is substantially under the control of gravity. A possible role of PsPINs and PsAUX1 of auxin polar transport in etiolated pea seedlings will also be discussed.

  9. 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.

  10. 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. PMID:12824397

  11. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

    PubMed

    Baines, Anthony J; Lu, Hui-Chun; Bennett, Pauline M

    2014-02-01

    Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and

  12. A Usual G-Protein-Coupled Receptor in Unusual Membranes.

    PubMed

    Chawla, Udeep; Jiang, Yunjiang; Zheng, Wan; Kuang, Liangju; Perera, Suchithranga M D C; Pitman, Michael C; Brown, Michael F; Liang, Hongjun

    2016-01-11

    G-protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and constitute about 50% of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge-interaction-directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid- and polymer-based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non-biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H(+)] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin-conserved ERY sequence motif that helps break the cytoplasmic "ionic lock". This study unveils a novel design concept of non-biological membranes to reconstitute and harness GPCR functions in synthetic systems. PMID:26633591

  13. Overcoming bottlenecks in the membrane protein structural biology pipeline.

    PubMed

    Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

    2016-06-15

    Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. PMID:27284049

  14. 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

  15. Membrane proteins of Mycoplasma bovis and their role in pathogenesis.

    PubMed

    Adamu, James Y; Wawegama, Nadeeka K; Browning, Glenn F; Markham, Philip F

    2013-10-01

    Mycoplasma membrane proteins influence cell shape, cell division, motility and adhesion to host cells, and are thought to be integrally involved in the pathogenesis of mycoplasmoses. Many of the membrane proteins predicted from mycoplasma genome sequences remain hypothetical, as their presence in cellular protein preparations is yet to be established experimentally. Recent genome sequences of several strains of Mycoplasma bovis have provided further insight into the potential role of the membrane proteins of this pathogen in colonisation and infection. This review highlights recent advances in knowledge about the influence of M. bovis membrane proteins on the pathogenesis of infection with this species and identifies future research directions for enhancing our understanding of the role of these proteins. PMID:23810376

  16. 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. PMID:25499533

  17. 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

    2011-05-01

    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. PMID:21476549

  18. Identification of genes encoding zinc finger proteins, non-histone chromosomal HMG protein homologue, and a putative GTP phosphohydrolase in the genome of Chilo iridescent virus.

    PubMed Central

    Schnitzler, P; Hug, M; Handermann, M; Janssen, W; Koonin, E V; Delius, H; Darai, C

    1994-01-01

    Five RNA transcripts of about 1.2 to 1.7 kilobases were mapped to a part of the genome of insect iridescent virus type 6 (Chilo iridescent virus; CIV) between genome coordinates 0.832 and 0.856 within the EcoRI DNA fragment F. The nucleotide sequence of this particular region (5702 base pairs) of the CIV genome was determined. The DNA sequence contains a number of perfect direct, inverted, and palindromic repeats including three clusters of tandemly organized repetitive DNA elements located between the nucleotide positions 1534 to 1566, 3720 to 3780, and 4350 to 4450. Eight long open reading frames (ORFs; EF1 to 8) were detected in the sequenced region of the CIV genome. ORF EF1 encodes a putative protein of 221 amino acid residues (aa) that is closely related to eukaryotic nonhistone chromosomal proteins of the high mobility group (HMG) superfamily. Virus encoded homologues of HMG proteins have not been reported so far. The EF2 gene product (145 aa) contains a specific zinc finger motif and belongs to a distinct group of identified and putative zinc finger proteins including a second putative protein (239 aa) of CIV encoded in the EcoRI DNA fragment Y (1984 bp; 0.381 to 0.391 viral map units). The product of EF6 (127 aa) is related to D250 ORF product of African swine fever virus (ASFV) and belongs to the recently described protein family sharing a highly conserved sequence motif with bacterial antimutator GTP phosphohydrolase MutT. Thus the sequenced region of the CIV genome encodes three putative proteins which may be directly involved in the replication and/or transcription of the viral DNA. Images PMID:8121799

  19. Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains.

    PubMed Central

    Kalus, Ina; Hodel, Alois; Koch, Annett; Kleene, Ralf; Edwardson, J Michael; Schrader, Michael

    2002-01-01

    Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin-GP-2 complex might play a role in signal transduction across the granule membrane. PMID:11853552

  20. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography

    PubMed Central

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A.; Wang, Dingjie; Liu, Wei; Spence, John C.H.; Doak, R. Bruce; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A.; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L.; Barty, Anton; Chapman, Henry N.; Kirian, Richard A.; Beyerlein, Kenneth R.; Stevens, Raymond C.; Li, Dianfan; Shah, Syed T.A.; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously-renewed source of material for serial femtosecond crystallography. Data collected from sub-10 μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor. PMID:24525480

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. Membrane Anchoring of Aminoacyl-tRNA Synthetases by Convergent Acquisition of a Novel Protein Domain*

    PubMed Central

    Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Ribas de Pouplana, Lluis; Luque, Ignacio

    2011-01-01

    Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain. PMID:21965654

  6. MALDI Tissue Profiling of Integral Membrane Proteins from Ocular Tissues

    PubMed Central

    Thibault, Danielle B.; Gillam, Christopher J.; Grey, Angus C.; Han, Jun; Schey, Kevin L.

    2008-01-01

    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 communication, 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 aged related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods. PMID:18396059

  7. Dynamic Nuclear Polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces

    PubMed Central

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

    2016-01-01

    We demonstrate that dynamic nuclear polarization (DNP) 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 6-fold for the dimeric protein. The enhancement affect 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. PMID:25828256

  8. G-protein-coupled glucocorticoid receptors on the pituitary cell membrane.

    PubMed

    Maier, Christina; Rünzler, Dominik; Schindelar, Julia; Grabner, Gottfried; Waldhäusl, Werner; Köhler, Gottfried; Luger, Anton

    2005-08-01

    Rapid, nongenomic actions of glucocorticoids (GCs) have been well documented, but information about putative membrane receptors that mediate them is scarce. We used fluorescence correlation spectroscopy to search for membrane GC-binding on the mouse pituitary cell line AtT-20. A slowly diffusing fraction (tau3; diffusion constant 3x10(-10) cm2 s-1) of fluorescein-labeled dexamethasone on the cell membrane corresponds to fluorescein-dexamethasone binding. Preincubation experiments were performed to test binding specificity: a 500-fold excess of unlabeled dexamethasone abolished subsequent fluorescein-dexamethasone membrane binding from 58+/-2 (control) to 8+/-1 (% of tau3, mean+/-s.e.m.), the natural ligand corticosterone prevented it partially (29+/-2), while the sex steroids estradiol (56+/-4) and progesterone (50+/-4) and the GC-receptor antagonist RU486 (56+/-2) had no effect. Preincubation with pertussis toxin resulted in disappearance of the slowest diffusion component (11+/-4) suggesting association of the receptor with a G-protein. Varying the concentration of fluorescein-dexamethasone showed that membrane binding is highly cooperative with an apparent Kd of 180 nM and Bmax of 230 nM. Taken together, these results demonstrate high-affinity GC-binding on the cell membrane of AtT-20 cells with characteristics distinct from intracellular binding. PMID:16079279

  9. Thermodynamics of protein driven self assembly in membranes

    NASA Astrophysics Data System (ADS)

    Natesan, Ramakrishnan; Tourdot, Richard; Bradley, Ryan; Radhakrishnan, Ravi

    2013-03-01

    Recent experimental evidences strongly point to the role of proteins and other membrane binding macromolecules in reshaping biological membranes, at length scales of the molecule and the structure enclosed by the membrane. In this work, we investigate the interplay between the membrane curvature induced at the molecular scale, mainly due to peripheral membrane proteins, and the resulting membrane morphologies, of varying complexity, observed at the mesoscale. The biological membrane, in our approach, is represented by a dynamically triangulated surface while the proteins are modeled as curvature fields on the membrane, which can either be isotropic or anisotropic. Thermal undulations in the membrane and cooperativity in the curvature field, due to the stabilization of a nematic phase, drives the membrane into conformations that resembles those in experiments in vivo and vitro. The stability of these structures are examined by two approaches to compute the free energy of the system: (i) Widom insertion technique to compute excess chemical potentials and (ii) thermodynamic integration using the Kirkwood coupling parameter to compute absolute free energies. Building on these methods, we propose a hybrid scheeme that couples both the approaches for computing free energies.

  10. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    PubMed Central

    Mourot, Brigitte; Nguyen, Thaovi; Fostier, Alexis; Bobe, Julien

    2006-01-01

    Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels. PMID:16457725

  11. Membrane Protein Production in the Yeast, S. cerevisiae.

    PubMed

    Cartwright, Stephanie P; Mikaliunaite, Lina; Bill, Roslyn M

    2016-01-01

    The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes. PMID:27485327

  12. Isolation and sequencing of a putative promoter region of the murine G protein beta 1 subunit (GNB1) gene.

    PubMed

    Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Wang, Xiao-Bing; Hembree, Cambria M; Goodman, Nancy L; Uhl, George R

    2002-02-01

    The expression of the heterotrimeric GTP-binding protein beta 1 subunit gene (GNB1) is regulated by psychostimulants such as cocaine and amphetamines. Since the up-regulation appears to be one of the candidate processes of sensitization, it is necessary to elucidate the cellular and molecular mechanism of the GNB1 gene regulation for a better understanding the establishment of sensitization. In the present study, we describe the isolation and nucleotide sequence analysis of the GNB1 gene promoter region. We have isolated approximately 10 kb of the 5'-flanking region of the mouse of GNB1 gene and found potential elements involved in putative transcriptional control of the GNB1, such as AP1, AP2, Sp1, cyclic AMP response element, and nuclear factor kappa B recognition sites, within the sequences 0.3 kb upstream from the putative transcription start site. This region was highly rich in G + C content, but lacked TATA or CATT boxes. Comparing the nucleotide sequence of the cDNA clone with the human genome databases using the BLAST program a region containing putative exon 1 and promoter of the human GNB1 gene in chromosome 1 was found. The cloning and sequence analysis of an extensive portion of the 5'-flanking regulatory region of the GNB1 gene provides new insights into the factors involved in the regulation by psychostimulants of GNB1 expression. PMID:12180136

  13. 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.

  14. 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. PMID:25817548

  15. Zonal rate model for stacked membrane chromatography part II: characterizing ion-exchange membrane chromatography under protein retention conditions.

    PubMed

    Francis, Patrick; von Lieres, Eric; Haynes, Charles

    2012-03-01

    The Zonal Rate Model (ZRM) has previously been shown to accurately account for contributions to elution band broadening, including external flow nonidealities and radial concentration gradients, in ion-exchange membrane (IEXM) chromatography systems operated under nonbinding conditions. Here, we extend the ZRM to analyze and model the behavior of retained proteins by introducing terms for intra-column mass transfer resistances and intrinsic binding kinetics. Breakthrough curve (BTC) data from a scaled-down anion-exchange membrane chromatography module using ovalbumin as a model protein were collected at flow rates ranging from 1.5 to 20 mL min(-1). Through its careful accounting of transport nonidealities within and external to the membrane stack, the ZRM is shown to provide a useful framework for characterizing putative protein binding mechanisms and models, for predicting BTCs and complex elution behavior, including the common observation that the dynamic binding capacity can increase with linear velocity in IEXM systems, and for simulating and scaling separations using IEXM chromatography. Global fitting of model parameters is used to evaluate the performance of the Langmuir, bi-Langmuir, steric mass action (SMA), and spreading-type protein binding models in either correlating or fundamentally describing BTC data. When combined with the ZRM, the bi-Langmuir, and SMA models match the chromatography data, but require physically unrealistic regressed model parameters to do so. In contrast, for this system a spreading-type model is shown to accurately predict column performance while also providing a realistic fundamental explanation for observed trends, including an observed increase in dynamic binding capacity with flow rate. PMID:22012741

  16. 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. PMID:26621472

  17. 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

  18. Membranes Do Not Tell Proteins How To Fold.

    PubMed

    Popot, Jean-Luc; Engelman, Donald M

    2016-01-12

    Which properties of the membrane environment are essential for the folding and oligomerization of transmembrane proteins? Because the lipids that surround membrane proteins in situ spontaneously organize into bilayers, it may seem intuitive that interactions with the bilayer provide both hydrophobic and topological constraints that help the protein to achieve a stable and functional three-dimensional structure. However, one may wonder whether folding is actually driven by the membrane environment or whether the folded state just reflects an adaptation of integral proteins to the medium in which they function. Also, apart from the overall transmembrane orientation, might the asymmetry inherent in biosynthesis processes cause proteins to fold to out-of-equilibrium, metastable topologies? Which of the features of a bilayer are essential for membrane protein folding, and which are not? To which extent do translocons dictate transmembrane topologies? Recent data show that many membrane proteins fold and oligomerize very efficiently in media that bear little similarity to a membrane, casting doubt on the essentiality of many bilayer constraints. In the following discussion, we argue that some of the features of bilayers may contribute to protein folding, stability and regulation, but they are not required for the basic three-dimensional structure to be achieved. This idea, if correct, would imply that evolution has steered membrane proteins toward an accommodation to biosynthetic pathways and a good fit into their environment, but that their folding is not driven by the latter or dictated by insertion apparatuses. In other words, the three-dimensional structure of membrane proteins is essentially determined by intramolecular interactions and not by bilayer constraints and insertion pathways. Implications are discussed. PMID:26649989

  19. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    SciTech Connect

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  20. Structure of a putative molybdenum-cofactor biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472)

    SciTech Connect

    Yoshida, Hiromi; Yamada, Mitsugu; Kuramitsu, Seiki; Kamitori, Shigehiro

    2008-07-01

    The crystal structure of a putative molybdenum-cofactor biosynthesis protein C (MoaC) from S. tokodaii (ST0472) was determined at 2.2 Å resolution. The crystal structure of a putative molybdenum-cofactor (Moco) biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472) was determined at 2.2 Å resolution. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 123.31, b = 78.58, c = 112.67 Å, β = 118.1°. The structure was solved by molecular replacement using the structure of Escherichia coli MoaC as the probe model. The asymmetric unit is composed of a hexamer arranged as a trimer of dimers with noncrystallographic 32 symmetry. The structure of ST0472 is very similar to that of E. coli MoaC; however, in the ST0472 protein an additional loop formed by the insertion of seven residues participates in intermonomer interactions and the new structure also reveals the formation of an interdimer β-sheet. These features may contribute to the stability of the oligomeric state.

  1. TMBETA-NET: discrimination and prediction of membrane spanning beta-strands in outer membrane proteins.

    PubMed

    Gromiha, M Michael; Ahmad, Shandar; Suwa, Makiko

    2005-07-01

    We have developed a web-server, TMBETA-NET for discriminating outer membrane proteins and predicting their membrane spanning beta-strand segments. The amino acid compositions of globular and outer membrane proteins have been systematically analyzed and a statistical method has been proposed for discriminating outer membrane proteins. The prediction of membrane spanning segments is mainly based on feed forward neural network and refined with beta-strand length. Our program takes the amino acid sequence as input and displays the type of the protein along with membrane-spanning beta-strand segments as a stretch of highlighted amino acid residues. Further, the probability of residues to be in transmembrane beta-strand has been provided with a coloring scheme. We observed that outer membrane proteins were discriminated with an accuracy of 89% and their membrane spanning beta-strand segments at an accuracy of 73% just from amino acid sequence information. The prediction server is available at http://psfs.cbrc.jp/tmbeta-net/. PMID:15980447

  2. Protein-driven membrane stresses in fusion and fission

    PubMed Central

    Kozlov, Michael M.; McMahon, Harvey T.; Chernomordik, Leonid V.

    2013-01-01

    Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cellsand release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and inmost cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling. PMID:20638285

  3. Association of Influenza Virus Proteins with Membrane Rafts

    PubMed Central

    Veit, Michael; Thaa, Bastian

    2011-01-01

    Assembly and budding of influenza virus proceeds in the viral budozone, a domain in the plasma membrane with characteristics of cholesterol/sphingolipid-rich membrane rafts. The viral transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) are intrinsically targeted to these domains, while M2 is seemingly targeted to the edge of the budozone. Virus assembly is orchestrated by the matrix protein M1, binding to all viral components and the membrane. Budding progresses by protein- and lipid-mediated membrane bending and particle scission probably mediated by M2. Here, we summarize the experimental evidence for this model with emphasis on the raft-targeting features of HA, NA, and M2 and review the functional importance of raft domains for viral protein transport, assembly and budding, environmental stability, and membrane fusion. PMID:22312341

  4. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

    PubMed

    Yamanaka, T; Ohta, T; Takahashi, M; Meshi, T; Schmidt, R; Dean, C; Naito, S; Ishikawa, M

    2000-08-29

    Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor. PMID:10944200

  5. Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

    PubMed Central

    Champion, C I; Blanco, D R; Exner, M M; Erdjument-Bromage, H; Hancock, R E; Tempst, P; Miller, J N; Lovett, M A

    1997-01-01

    In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum. PMID:9023206

  6. Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes

    PubMed Central

    Im, Hyungsoon; Wittenberg, Nathan J.; Lesuffleur, Antoine; Lindquist, Nathan C.; Oh, Sang-Hyun

    2010-01-01

    Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with α-hemolysin (α-HL). The incorporation of α-HL into the membrane followed by specific antibody binding (anti-α-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that α-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes. PMID:21218136

  7. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    PubMed Central

    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. PMID:26771082

  8. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Champion, C I; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1996-01-01

    The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete. PMID:8759855

  9. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

    PubMed

    Baker, Lindsay A; Daniëls, Mark; van der Cruijsen, Elwin A W; Folkers, Gert E; Baldus, Marc

    2015-06-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR. PMID:25956570

  10. Expression of sarcomeric proteins and assembly of myofibrils in the putative myofibroblast cell line BHK-21/C13.

    PubMed

    van der Ven, P F; Fürst, D O

    1998-10-01

    The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins. PMID:9836147

  11. Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry.

    PubMed

    Cathro, Peter; McCarthy, Peter; Hoffmann, Peter; Zilm, Peter

    2016-06-01

    Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC-ESI mass spectrometry. We identified 222 membrane-associated proteins, which represent approximately 24% of the predicted membrane-associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane-associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment. PMID:27419061

  12. 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.

  13. Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

    PubMed Central

    McGovern, K; Ehrmann, M; Beckwith, J

    1991-01-01

    We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them. Images PMID:1915262

  14. Organization and dynamics of SNARE proteins in the presynaptic membrane

    PubMed Central

    Milovanovic, Dragomir; Jahn, Reinhard

    2015-01-01

    Our view of the lateral organization of lipids and proteins in the plasma membrane has evolved substantially in the last few decades. It is widely accepted that many, if not all, plasma membrane proteins and lipids are organized in specific domains. These domains vary widely in size, composition, and stability, and they represent platforms governing diverse cell functions. The presynaptic plasma membrane is a well-studied example of a membrane which undergoes rearrangements, especially during exo- and endocytosis. Many proteins and lipids involved in presynaptic function are known, and major efforts have been made to understand their spatial organization and dynamics. Here, we focus on the mechanisms underlying the organization of SNAREs, the key proteins of the fusion machinery, in distinct domains, and we discuss the functional significance of these clusters. PMID:25852575

  15. Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

    PubMed

    Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

    2016-01-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

  16. The contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in primary hypotension.

    PubMed

    Ivanov, V P; Polonikov, A V; Solodilova, M A

    2005-01-01

    Our previous studies have shown that, compared with healthy individuals, patients with primary arterial hypotension (PAH) have significant quantitative changes in erythrocyte membrane proteins. The purpose of the present study was to evaluate the contribution made by genetic and environmental factors to quantitative variation of erythrocyte membrane proteins in PAH. We studied 109 hypotensive patients, 124 normotensive subjects, 222 of their first-degree relatives and 24 twin pairs by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The decomposition of total phenotypic variance of erythrocyte membrane proteins to genetic and environmental components was performed on the basis of correlations among first-degree relatives by the least squares method. The genetic dominance and shared environmental factors were found to influence the variability of cytoskeletal membrane proteins whose contents were changed in PAH. Furthermore, variations in alpha-spectrin, actin and anion exchanger in hypotensives were substantially influenced by major gene and maternal effects. Ankyrin 2.1 and actin content was under the control of common underlying genes. Variations in membrane-associated glutathione-S-transferase and tropomyosin were predominantly affected by polygenes. These findings suggest that the putative major genes with pleiotropic effects appear to be involved in the control of quantitative disorders of erythrocyte membrane proteins in primary hypotension. PMID:15638825

  17. 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.

  18. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

    Radoicic, Jasmina; Lu, George J.; Opella, Stanley J.

    2014-01-01

    Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function. PMID:25032938

  19. 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

  20. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    NASA Astrophysics Data System (ADS)

    Sun, Junfen; Wu, Lishun

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  1. Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum.

    PubMed Central

    Hagman, K E; Porcella, S F; Popova, T G; Norgard, M V

    1997-01-01

    The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the

  2. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  3. Exceptional overproduction of a functional human membrane protein.

    PubMed

    Nyblom, Maria; Oberg, Fredrik; Lindkvist-Petersson, Karin; Hallgren, Karin; Findlay, Heather; Wikström, Jennie; Karlsson, Anders; Hansson, Orjan; Booth, Paula J; Bill, Roslyn M; Neutze, Richard; Hedfalk, Kristina

    2007-11-01

    Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. PMID:17869538

  4. Phytochemicals perturb membranes and promiscuously alter protein function.

    PubMed

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  5. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  6. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  7. Clipping or Extracting: Two Ways to Membrane Protein Degradation.

    PubMed

    Avci, Dönem; Lemberg, Marius K

    2015-10-01

    Protein degradation is a fundamentally important process that allows cells to recognize and remove damaged protein species and to regulate protein abundance according to functional need. A fundamental challenge is to understand how membrane proteins are recognized and removed from cellular organelles. While most of our understanding of this mechanism comes from studies on p97/Cdc48-mediated protein dislocation along the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, recent studies have revealed intramembrane proteolysis to be an additional mechanism that can extract transmembrane segments. Here, we review these two principles in membrane protein degradation and discuss how intramembrane proteolysis, which introduces an irreversible step in protein dislocation, is used to drive regulated protein turnover. PMID:26410407

  8. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  9. Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

    PubMed Central

    Burns, C C; Poss, M L; Thomas, E; Overbaugh, J

    1995-01-01

    A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity. PMID:7884859

  10. Active membrane transport and receptor proteins from bacteria.

    PubMed

    Saidijam, M; Bettaney, K E; Szakonyi, G; Psakis, G; Shibayama, K; Suzuki, S; Clough, J L; Blessie, V; Abu-Bakr, A; Baumberg, S; Meuller, J; Hoyle, C K; Palmer, S L; Butaye, P; Walravens, K; Patching, S G; O'reilly, J; Rutherford, N G; Bill, R M; Roper, D I; Phillips-Jones, M K; Henderson, P J F

    2005-08-01

    A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. PMID:16042616

  11. Domain formation in membranes caused by lipid wetting of protein.

    PubMed

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data. PMID:18643096

  12. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  13. The functions of tryptophan residues in membrane proteins

    SciTech Connect

    Schiffer, M.; Chang, C.H.; Stevens, F.J.

    1994-08-01

    Membrane proteins in general have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are located mostly in the segments that connect the transmembrane helices. Further, they are concentrated at the periplasmic side of the complex. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. We suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.

  14. Mechanisms of integral membrane protein insertion and folding

    PubMed Central

    2014-01-01

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. PMID:25277655

  15. Predictive energy landscapes for folding membrane protein assemblies

    NASA Astrophysics Data System (ADS)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  16. Membrane protein properties revealed through data-rich electrostatics calculations

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

    2015-01-01

    SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

  17. Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium.

    PubMed

    Chung, I; Burkart, A; Szmydynger-Chodobska, J; Dodd, K A; Trimble, W S; Miller, K V; Shim, M; Chodobski, A

    2003-01-01

    In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of

  18. Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis.

    PubMed Central

    Sommer, P; Gleyzal, C; Guerret, S; Etienne, J; Grimaud, J A

    1992-01-01

    There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies. Images PMID:1530927

  19. The putative cell cycle gene, enhancer of rudimentary, encodes a highly conserved protein found in plants and animals.

    PubMed

    Gelsthorpe, M; Pulumati, M; McCallum, C; Dang-Vu, K; Tsubota, S I

    1997-02-28

    The enhancer of rudimentary gene, e(r), in Drosophila melanogaster encodes a protein, ER, whose function has been implicated in pyrimidine biosynthesis and the cell cycle (Wojcik et al. (1994) Genetics 138, 1163-1170). In order to identify conserved regions of the protein and potentially important functional domains, the e(r) gene was cloned and sequenced from two other insects (Drosophila virilis and Aedes aegypti) and three vertebrates (Homo sapiens, Mus musculus, and Brachydanio rerio) and sequenced from a flowering plant (Arabidopsis thaliana). These sequences along with those of a nematode (Caenorhabditis elegans) exhibit a high degree of identity. ER of Drosophila melanogaster is 76% identical to the three vertebrate proteins, 49% identical to the nematode protein, and 40% identical to the plant protein. There is high evolutionary conservation among the vertebrates. The mouse and human proteins are identical and differ from that of the zebrafish by a single conservative amino-acid change (valine for isoleucine). A dramatic sequence conservation is seen in the position of the hydrophobic amino acids. Of the 27 positions occupied by hydrophobic amino acids in ER of Drosophila melanogaster, 25 of the corresponding positions in the human protein, 23 of the positions in Caenorhabditis elegans, and 20 of the positions in Arabidopsis thaliana have hydrophobic amino acids. Most of these residues are present in three conserved amphipathic alpha-helices, which are proposed to function in protein-protein interactions. Two phosphorylation sites for casein kinase II (CKII) have also been conserved within the animal groups. Purified ER from Drosophila melanogaster is phosphorylated in vitro by CKII, arguing that these two sites are functional in vivo. A putative shift in the secondary structure of ER caused by the phosphorylation of these sites suggests that CKII may be regulating the activity of the ER in vivo. PMID:9074495

  20. Setting up and running molecular dynamics simulations of membrane proteins.

    PubMed

    Kandt, Christian; Ash, Walter L; Tieleman, D Peter

    2007-04-01

    Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins. PMID:17367719

  1. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  2. Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

    PubMed Central

    Sanz-Hernández, Máximo; Vostrikov, Vitaly V.; Veglia, Gianluigi; De Simone, Alfonso

    2016-01-01

    The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states. PMID:26975211

  3. The putative HORMA domain protein Atg101 dimerizes and is required for starvation-induced and selective autophagy in Drosophila.

    PubMed

    Hegedűs, Krisztina; Nagy, Péter; Gáspári, Zoltán; Juhász, Gábor

    2014-01-01

    The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes. PMID:24895579

  4. Membrane bending by protein crowding is affected by protein lateral confinement.

    PubMed

    Derganc, Jure; Čopič, Alenka

    2016-06-01

    Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches. PMID:26969088

  5. Structural adaptations of proteins to different biological membranes

    PubMed Central

    Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

    2013-01-01

    To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

  6. Quality control of nonstop membrane proteins at the ER membrane and in the cytosol.

    PubMed

    Arakawa, Shunsuke; Yunoki, Kaori; Izawa, Toshiaki; Tamura, Yasushi; Nishikawa, Shuh-Ichi; Endo, Toshiya

    2016-01-01

    Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions. PMID:27481473

  7. Quality control of nonstop membrane proteins at the ER membrane and in the cytosol

    PubMed Central

    Arakawa, Shunsuke; Yunoki, Kaori; Izawa, Toshiaki; Tamura, Yasushi; Nishikawa, Shuh-ichi; Endo, Toshiya

    2016-01-01

    Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions. PMID:27481473

  8. The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

    PubMed Central

    Overmeyer, Jean H.; Wilson, Amy L.; Erdman, Robert A.; Maltese, William A.

    1998-01-01

    Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate. PMID:9437002

  9. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    PubMed

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  10. Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

    PubMed Central

    Tonnesen, Asger; Christensen, Sune M.; Tkach, Vadym; Stamou, Dimitrios

    2014-01-01

    Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm−1) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 kBT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general. PMID:24411252

  11. Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

    SciTech Connect

    Straatsma, TP

    2006-12-01

    Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

  12. [Multiparticle computer simulation of protein interactions in the photosynthetic membrane].

    PubMed

    Riznichenko, G Iu; Kovalenko, I B; Abaturova, A M; D'iakonova, A N; Kniazeva, O S; Ustinin, D M; Khrushchev, S S; Rubin, A B

    2011-01-01

    The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the protein plastocyanin with the protein cytochrome f and the pigment-protein complex of photosystem I, as well as of the protein ferredoxin with the protein FNR and photosystem 1 are considered. The role of diffusion and electrostatic interactions is discussed, and the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed. PMID:22117434

  13. Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the Tyrosine O-Glycosylation System of Paenibacillus alvei CCM 2051T

    PubMed Central

    Ristl, Robin; Janesch, Bettina; Anzengruber, Julia; Forsthuber, Agnes; Blaha, Johanna; Messner, Paul; Schäffer, Christina

    2015-01-01

    Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy_C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate

  14. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  15. Encapsulated membrane proteins: A simplified system for molecular simulation.

    PubMed

    Lee, Sarah C; Khalid, Syma; Pollock, Naomi L; Knowles, Tim J; Edler, Karen; Rothnie, Alice J; R T Thomas, Owen; Dafforn, Timothy R

    2016-10-01

    Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946242

  16. A putative precursor protein in the evolution of the bean alpha-amylase inhibitor.

    PubMed

    Finardi-Filho, F; Mirkov, T E; Chrispeels, M J

    1996-09-01

    Seeds of the common bean Phaseolus vulgaris and the tepary bean (P. acutifolius) contain a family of plant defence proteins that includes phytohaemagglutinin (PHA), arcelin and alpha-amylase inhibitor (alpha AI). These homologous proteins differ by the absence of short loops at the surface of the protein and by the presence of a proteolytic processing site (Asn77) that allows alpha AI to be post-translationally cleaved and activated. We now report the derived amino acid sequence of two amylase inhibitor-like (AIL) proteins that are not proteolytically processed, although they have the typical processing site. One protein is from the common bean, and the other from the tepary bean. On a dendrogram, these proteins are grouped with alpha AIs rather than with the arcelins or lectins. alpha AI differs from AIL primarily by the deletion of a 15-amino-acid segment from the middle of the AIL sequence. When alpha AI is expressed in tobacco, it is proteolytically processed to form an active molecule. However, AIL sequences are not processed. We suggest that the AIL proteins may be an intermediate in the evolution of an active alpha AI. PMID:8987505

  17. Swimbladder membrane protein of an abyssal fish, Coryphaenoides acrolepis.

    PubMed

    Mosholder, R S; Josephson, R V; Phleger, C F

    1979-01-01

    Protein components of the membranous foamy tissue collected from the swimbladder of Coryphaenoides acrolepis, a continental slope/deep sea grenadier fish, were partially fractionated and characterized by procedures used successfully for erythrocyte membrane proteins. Methods involving pH and ionic strength adjustment in the presence of EDTA and beta-mercaptoethanol resulted in some protein fractionation but no distinct separation or isolation of membrane proteins. Gel filtration by Sephadex G-100 and Sepharose 2B in the presence of dodecyl sulfate partially fractionated protein species between 18,000 and 150,000 molecular weight (as confirmed by dodecyl sulfate polyacrylamide gel electrophoresis). Low molecular weight proteins were resolvable into a few diffuse and streaky bands by dodecyl sulfate and chloral hydrate polyacrylamide gel electrophoresis, the former giving superior reso-ution. A major fraction of large molecular weight protein (greater than or equal to 40 X 10(6)) was not resolved by any method. A possible explanation for these unusual findings is that decompression due to rapid ascent of the fish from deep ocean caused irreversible alteration of swimbladder membrane proteins. PMID:504363

  18. [Elution of urinary proteins preserved on nitrocellulose membrane with heating].

    PubMed

    Qin, Weiwei; Gao, Youhe

    2015-09-01

    The preservation of urinary proteins on a membrane plays a vital role in biomarker research, and the efficient elution of proteins preserved on nitrocellulose membrane (NC membrane) determines the application of this method. During the heating elution procedure, we raised the temperature to reduce the intense vortexing time, and kept gentle rotating while precipitation to prevent nitrocellulose reformation. We also used SDS-PAGE and LC-MS/MS to analyze the urinary proteins prepared by heating elution procedure, intense vortexing elution procedure and acetone precipitation method. There was no degradation of proteins prepared by heating elution procedure. Compared with proteins prepared by heating elution method and acetone precipitation method, the overlapping rates of the proteins was almost the same (92.6% versus 96.8%) and the ratios of CV values (< 20%) of the proteins were both high (85.2% and 94.4%). The heating elution procedure achieved good technical reproducibility, and was much simpler and more efficient than the previous one. It can facilitate the application of the preservation of urinary proteins on membrane. PMID:26955716

  19. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  20. Targeting membrane proteins for antibody discovery using phage display

    PubMed Central

    Jones, Martina L.; Alfaleh, Mohamed A.; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W.; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B.; Chin, David Y.; Mahler, Stephen M.

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  1. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    NASA Astrophysics Data System (ADS)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  2. Targeting membrane proteins for antibody discovery using phage display.

    PubMed

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  3. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-06-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. PMID:3380805

  4. Ultrafast permeation of water through protein-based membranes.

    PubMed

    Peng, Xinsheng; Jin, Jian; Nakamura, Yoshimichi; Ohno, Takahisa; Ichinose, Izumi

    2009-06-01

    Pressure-driven filtration by porous membranes is widely used in the production of drinking water from ground and surface water. Permeation theory predicts that filtration rate is proportional to the pressure difference across the filtration membrane and inversely proportional to the thickness of the membrane. However, these membranes need to be able to withstand high water fluxes and pressures, which means that the active separation layers in commercial filtration systems typically have a thickness of a few tens to several hundreds of nanometres. Filtration performance might be improved by the use of ultrathin porous silicon membranes or carbon nanotubes immobilized in silicon nitride or polymer films, but these structures are difficult to fabricate. Here, we report a new type of filtration membrane made of crosslinked proteins that are mechanically robust and contain channels with diameters of less than 2.2 nm. We find that a 60-nm-thick membrane can concentrate aqueous dyes from fluxes up to 9,000 l h(-1) m(-2) bar(-1), which is approximately 1,000 times higher than the fluxes that can be withstood by commercial filtration membranes with similar rejection properties. Based on these results and molecular dynamics simulations, we propose that protein-surrounded channels with effective lengths of less than 5.8 nm can separate dye molecules while allowing the ultrafast permeation of water at applied pressures of less than 1 bar. PMID:19498395

  5. Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein.

    PubMed

    Singh, Alla; Ubaid-Ullah, Shah; Batra, Janendra K

    2016-09-01

    RNase P is involved in processing the 5' end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The Km of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation. PMID:27417238

  6. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  7. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  8. Cataloging proteins putatively secreted during the biotrophy-necrotrophy transition of the anthracnose pathogen Colletotrichum truncatum.

    PubMed

    Bhadauria, Vijai; Banniza, Sabine; Vandenberg, Albert; Selvaraj, Gopalan; Wei, Yangdou

    2011-10-01

    Hemibiotrophic phytopathogenic fungi cause devastating diseases in agronomically important crops. These fungal pathogens exploit a stealth bi-phasic infection strategy to colonize host plants. Their morphological and nutritional transition from biotrophy (characterized by voluminous intracellular primary hyphae) to necrotrophy (characterized by thin secondary hyphae) known as the biotrophy-necrotrophy switch (hemibiotrophy) is critical in symptom and disease development. To establish successful hemibiotrophic parasitism, pathogens likely secrete suites of proteins at the switch that constitute the biotrophy-necrotrophy switch secretome. To catalogue such proteins, a directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the switch of Colletotrichum truncatum, and 5000 expressed sequence tags (ESTs) were generated. Four potential groups (hydrolytic enzymes, cell envelope-associated proteins [CEAPs], candidate effectors and proteins with diverse functions) were identified from pathogen-derived ESTs. Expression profiling of transcripts encoding CEAPs and candidate effectors in an infection time-course revealed that the majority of these transcripts were expressed or induced during the necrotrophic phase and repressed during the biotrophic phase of in planta colonization, indicating the massive accumulation of proteins at the switch. Taken together, our data suggest that the hemibiotrophic mode of fungal proliferation entails complex interactions of a pathogen with its host wherein the pathogen requires live host cells prior to switching to the necrotrophic phase. The microbial proteins employed during pathogenesis are likely to have defined roles at specific stages of pathogenesis. PMID:21897125

  9. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  10. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  11. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

    PubMed Central

    Kropinski, A M; Parr, T R; Angus, B L; Hancock, R E; Ghiorse, W C; Greenberg, E P

    1987-01-01

    The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. Images PMID:3025168

  12. Quality control of integral membrane proteins by assembly-dependent membrane integration.

    PubMed

    Feige, Matthias J; Hendershot, Linda M

    2013-08-01

    Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis. PMID:23932713

  13. Lipids assist the membrane insertion of a BAM-independent outer membrane protein

    PubMed Central

    Huysmans, Gerard H. M.; Guilvout, Ingrid; Chami, Mohamed; Nickerson, Nicholas N.; Pugsley, Anthony P.

    2015-01-01

    Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved β-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent. PMID:26463896

  14. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    PubMed Central

    2011-01-01

    Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs) were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8). The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are metal-dependent hydrolases

  15. Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport.

    PubMed

    Soneoka, Y; Kingsman, S M; Kingsman, A J

    1997-07-01

    We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions. PMID:9188629

  16. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  17. A new window into the molecular physiology of membrane proteins

    PubMed Central

    Landreh, Michael; Robinson, Carol V

    2015-01-01

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome. PMID:25630257

  18. A new window into the molecular physiology of membrane proteins.

    PubMed

    Landreh, Michael; Robinson, Carol V

    2015-01-15

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome. PMID:25630257

  19. Phagocyte-derived S100 proteins in autoinflammation: putative role in pathogenesis and usefulness as biomarkers.

    PubMed

    Kessel, Christoph; Holzinger, Dirk; Foell, Dirk

    2013-06-01

    The cytoplasmic S100 proteins derived from cells of myeloid origin are promising new markers of (auto-)inflammation. S100A8/A9 and S100A12 are released from monocytes and granulocytes during activation of the innate immune system. Tissue and serum concentrations correlate to disease activity, both during local and systemic inflammation. In autoinflammatory diseases such as Familial Mediterranean Fever (FMF) and Systemic onset Juvenile Idiopathic Arthritis (SJIA), a dysregulation of alternative secretory pathways may be involved in pathogenesis and lead to hypersecretion of S100 proteins. Since autoinflammatory diseases can be difficult to diagnose, phagocyte-derived S100 proteins are valid tools in the diagnosis of autoinflammatory diseases. In addition, they may help achieve a better understanding of the pathophysiology of autoinflammatory disorders including SJIA and FMF, and even provide novel therapeutic targets in the future. PMID:23269200

  20. 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.

  1. Protein Z: A putative novel biomarker for early detection of ovarian cancer.

    PubMed

    Russell, Matthew R; Walker, Michael J; Williamson, Andrew J K; Gentry-Maharaj, Aleksandra; Ryan, Andy; Kalsi, Jatinderpal; Skates, Steven; D'Amato, Alfonsina; Dive, Caroline; Pernemalm, Maria; Humphryes, Phillip C; Fourkala, Evangelia-Ourania; Whetton, Anthony D; Menon, Usha; Jacobs, Ian; Graham, Robert L J

    2016-06-15

    Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down-regulated up to 2 years pre-diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up-regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA-125 and CA-125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA-125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes. PMID:26815306

  2. Protein Z: A putative novel biomarker for early detection of ovarian cancer

    PubMed Central

    Russell, Matthew R.; Walker, Michael J.; Williamson, Andrew J. K.; Gentry‐Maharaj, Aleksandra; Ryan, Andy; Kalsi, Jatinderpal; Skates, Steven; D'Amato, Alfonsina; Dive, Caroline; Pernemalm, Maria; Humphryes, Phillip C.; Fourkala, Evangelia‐Ourania; Whetton, Anthony D.; Menon, Usha

    2016-01-01

    Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes. PMID:26815306

  3. Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

    PubMed

    Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

    2016-09-01

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger. PMID:27088852

  4. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

    PubMed Central

    2013-01-01

    Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence

  5. Molecular Characterization of the Mycoplasma gallisepticum pvpA Gene Which Encodes a Putative Variable Cytadhesin Protein

    PubMed Central

    Boguslavsky, S.; Menaker, D.; Lysnyansky, I.; Liu, T.; Levisohn, S.; Rosengarten, R.; García, M.; Yogev, D.

    2000-01-01

    A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum. PMID:10858209

  6. Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein.

    PubMed

    Boguslavsky, S; Menaker, D; Lysnyansky, I; Liu, T; Levisohn, S; Rosengarten, R; García, M; Yogev, D

    2000-07-01

    A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum. PMID:10858209

  7. Putative rust fungal effector proteins in infected bean and soybean leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but the effector repertoire for U. appendiculatus and P. pachyrhizi is not fully def...

  8. LINGO-1 Protein Interacts with the p75 Neurotrophin Receptor in Intracellular Membrane Compartments*

    PubMed Central

    Meabon, James S.; De Laat, Rian; Ieguchi, Katsuaki; Wiley, Jesse C.; Hudson, Mark P.; Bothwell, Mark

    2015-01-01

    Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75NTR·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75NTR associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75NTR. Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75NTR in a manner that is difficult to reconcile with the existence of a LINGO-1·p75NTR·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75NTR·NgR complexes in the plasma membrane. PMID:25666623

  9. A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties.

    PubMed

    Tynyakov, Jenny; Bentov, Shmuel; Abehsera, Shai; Yehezkel, Galit; Roth, Ziv; Khalaila, Isam; Weil, Simy; Berman, Amir; Plaschkes, Inbar; Tom, Moshe; Aflalo, Eliahu D; Sagi, Amir

    2015-11-01

    Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers-Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was also found to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation. PMID:26385331

  10. Photolabeling of brain membrane proteins by lysergic acid diethylamide.

    PubMed

    Mahon, A C; Hartig, P R

    1982-04-01

    3H-Lysergic acid diethylamide (3H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a subpopulation of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays. PMID:7087658

  11. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  12. Reconstructing protein remodeled membranes in molecular detail from mesoscopic models

    PubMed Central

    Lyman, Edward; Cui, Haosheng; Voth, Gregory A.

    2014-01-01

    We present a method for “inverse coarse graining,” rebuilding a higher resolution model from a lower resolution one, in order to rebuild protein coats for remodeled membranes of complex topology. The specific case of membrane remodeling by N-BAR domain containing proteins is considered here, although the overall method is general and thus applicable to other membrane remodeling phenomena. Our approach begins with a previously developed, discretized mesoscopic continuum membrane model (EM2) which has been shown to capture the reticulated membrane topologies often observed for N-BAR/liposome systems by electron microscopy (EM). The information in the EM2 model — directions of the local curvatures and a low resolution sample of the membrane surface — is then used to construct a coarse-grained (CG) system with one site per lipid and 26 sites per protein. We demonstrate the approach on pieces of EM2 structures with three different topologies that have been observed by EM: A tubule, a “Y” junction, and a torus. We show that the approach leads to structures that are stable under subsequent constant temperature CG simulation, and end by considering the future application of the methodology as a hybrid approach that combines experimental information with computer modeling. PMID:21503332

  13. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    PubMed

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling. PMID:27088673

  14. Dynamic Regulation of Mammalian Numb by G Protein-coupled Receptors and Protein Kinase C Activation: Structural Determinants of Numb Association with the Cortical Membrane

    PubMed Central

    Dho, Sascha E.; Trejo, JoAnn; Siderovski, David P.

    2006-01-01

    The cell fate determinant Numb is a membrane-associated adaptor protein involved in both development and intracellular vesicular trafficking. It has a phosphotyrosine-binding (PTB) domain and COOH-terminal endocytic-binding motifs for α-adaptin and Eps15 homology domain-containing proteins. Four isoforms of Numb are expressed in vertebrates, two of which selectively associate with the cortical membrane. In this study, we have characterized a cortical pool of Numb that colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. Green fluorescent protein (GFP)-tagged mutants of Numb were used to identify the structural determinants required for localization. In addition to the previously described association of the PTB domain with the plasma membrane, we show that the AP2-binding motifs facilitate the association of Numb with cortical membrane punctae and vesicles. We also show that agonist stimulation of G protein-coupled receptors (GPCRs) that are linked to phospholipase Cβ and protein kinase C (PKC) activation causes redistribution of Numb from the cortical membrane to the cytosol. This effect is correlated with Numb phosphorylation and an increase in its Triton X-100 solubility. Live-imaging analysis of mutants identified two regions within Numb that are independently responsive to GPCR-mediated lipid hydrolysis and PKC activation: the PTB domain and a region encompassing at least three putative PKC phosphorylation sites. Our data indicate that membrane localization of Numb is dynamically regulated by GPCR-activated phospholipid hydrolysis and PKC-dependent phosphorylation events. PMID:16837553

  15. PARAQUAT RESISTANT1, a Golgi-Localized Putative Transporter Protein, Is Involved in Intracellular Transport of Paraquat1[C][W

    PubMed Central

    Li, Jianyong; Mu, Jinye; Bai, Jiaoteng; Fu, Fuyou; Zou, Tingting; An, Fengying; Zhang, Jian; Jing, Hongwei; Wang, Qing; Li, Zhen; Yang, Shuhua; Zuo, Jianru

    2013-01-01

    Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis (Arabidopsis thaliana) mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to a reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative l-type amino acid transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast and also provide a practical approach for genetic manipulations of paraquat resistance in crops. PMID:23471133

  16. Role of lipids in the translocation of proteins across membranes.

    PubMed Central

    Van Voorst, F; De Kruijff, B

    2000-01-01

    The architecture of cells, with various membrane-bound compartments and with the protein synthesizing machinery confined to one location, dictates that many proteins have to be transported through one or more membranes during their biogenesis. A lot of progress has been made on the identification of protein translocation machineries and their sorting signals in various organelles and organisms. Biochemical characterization has revealed the functions of several individual protein components. Interestingly, lipid components were also found to be essential for the correct functioning of these translocases. This led to the idea that there is a very intimate relationship between the lipid and protein components that enables them to fulfil their intriguing task of transporting large biopolymers through a lipid bilayer without leaking their contents. In this review we focus on the Sec translocases in the endoplasmic reticulum and the bacterial inner membrane. We also highlight the interactions of lipids and proteins during the process of translocation and integrate this into a model that enables us to understand the role of membrane lipid composition in translocase function. PMID:10769162

  17. Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies.

    PubMed

    Ehsan, Muhammad; Du, Yang; Scull, Nicola J; Tikhonova, Elena; Tarrasch, Jeffrey; Mortensen, Jonas S; Loland, Claus J; Skiniotis, Georgios; Guan, Lan; Byrne, Bernadette; Kobilka, Brian K; Chae, Pil Seok

    2016-03-23

    Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins. PMID:26966956

  18. An improved tripod amphiphile for membrane protein solubilization.

    PubMed Central

    Yu, S. M.; McQuade, D. T.; Quinn, M. A.; Hackenberger, C. P.; Krebs, M. P.; Polans, A. S.; Gellman, S. H.

    2000-01-01

    Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a "tripod amphiphile," that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal. PMID:11206073

  19. Phosphoproteins and protein kinases of the Golgi apparatus membrane

    SciTech Connect

    Capasso, J.M.; Abeijon, C.; Hirschberg, C.B.

    1985-11-25

    Incubation of a highly purified fraction derived from rat liver Golgi apparatus with (gamma-TSP)ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on MgS , independent of CaS , calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on CaS and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.

  20. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  1. Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

    PubMed

    Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

    2001-06-19

    Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research. PMID:11416223

  2. The putative multidrug resistance protein MRP-7 inhibits methylmercury-associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans.

    PubMed

    VanDuyn, Natalia; Nass, Richard

    2014-03-01

    Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene-environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill-defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here, we report that a gene coding for the putative multidrug resistance protein MRP-7 in Caenorhabditis elegans modulates whole animal and DA neuron sensitivity to MeHg. In this study, we demonstrate that genetic knockdown of MRP-7 results in a twofold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg-associated animal death. Chronic exposure to low concentrations of MeHg induces MRP-7 gene expression, while exposures in MRP-7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP-7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg-associated DA neuron pathology. PMID:24266639

  3. Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

    PubMed

    Humbert, María Victoria; Hung, Miao-Chiu; Phillips, Renee; Akoto, Charlene; Hill, Alison; Tan, Wei-Ming; Heckels, John Edward; Christodoulides, Myron

    2016-01-01

    The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3-14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3-14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the h

  4. Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis

    PubMed Central

    Akoto, Charlene; Hill, Alison; Tan, Wei-Ming; Heckels, John Edward; Christodoulides, Myron

    2016-01-01

    The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the h

  5. Conserved Determinants for Membrane Association of Nonstructural Protein 5A from Hepatitis C Virus and Related Viruses▿

    PubMed Central

    Brass, Volker; Pal, Zsuzsanna; Sapay, Nicolas; Deléage, Gilbert; Blum, Hubert E.; Penin, François; Moradpour, Darius

    2007-01-01

    Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation. PMID:17192310

  6. Regulation of protein mobility via thermal membrane undulations.

    PubMed

    Brown, Frank L H

    2003-02-01

    The in-plane diffusivelike motion of membrane bound proteins on the surface of cells is considered. We suggest, on the basis of theoretical arguments and simulation, that thermally excited undulations of the lipid bilayer may serve as a mechanism for proteins to hop between adjacent regions on the cell surface separated by barriers composed of internal cellular structure (e.g., the cytoskeleton). We specifically investigate the mobility of band 3 dimer on the surface of red blood cells where the spectrin cytoskeletal meshwork defines a series of "corrals" on the cell surface known to hinder protein motion. Previous models of this system have postulated that the cytoskeleton must deform to allow passage of membrane bound proteins out of these corral regions and have ignored fluctuations of the bilayer. Our model provides a complementary mechanism and we posit that the mobility of real proteins in real cells is likely the result of several mechanisms acting in parallel. PMID:12547768

  7. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted. PMID:24928741

  8. A pollen-specific calmodulin-binding protein, NPG1, interacts with putative pectate lyases

    PubMed Central

    Shin, Sung-Bong; Golovkin, Maxim; Reddy, Anireddy S. N.

    2014-01-01

    Previous genetic studies have revealed that a pollen-specific calmodulin-binding protein, No Pollen Germination 1 (NPG1), is required for pollen germination. However, its mode of action is unknown. Here we report direct interaction of NPG1 with pectate lyase-like proteins (PLLs). A truncated form of AtNPG1 lacking the N-terminal tetratricopeptide repeat 1 (TPR1) failed to interact with PLLs, suggesting that it is essential for NPG1 interaction with PLLs. Localization studies with AtNPG1 fused to a fluorescent reporter driven by its native promoter revealed its presence in the cytosol and cell wall of the pollen grain and the growing pollen tube of plasmolyzed pollen. Together, our data suggest that the function of NPG1 in regulating pollen germination is mediated through its interaction with PLLs, which may modify the pollen cell wall and regulate pollen tube emergence and growth. PMID:24919580

  9. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    SciTech Connect

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-09-27

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.

  10. Expressing and purifying membrane transport proteins in high yield.

    PubMed

    Hale, Calvin C; Hill, Chananada K; Price, Elmer M; Bossuyt, Julie

    2002-01-01

    Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic. PMID:11741710

  11. Identification of outer membrane proteins of Mycobacterium tuberculosis.

    PubMed

    Song, Houhui; Sandie, Reatha; Wang, Ying; Andrade-Navarro, Miguel A; Niederweis, Michael

    2008-11-01

    The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis. PMID:18439872

  12. Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

    NASA Technical Reports Server (NTRS)

    Baird, R. A.; Steyger, P. S.; Schuff, N. R.

    1997-01-01

    Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.

  13. The C. elegans sex-determining gene fem-2 encodes a putative protein phosphatase.

    PubMed Central

    Pilgrim, D; McGregor, A; Jäckle, P; Johnson, T; Hansen, D

    1995-01-01

    The genetic and molecular analysis of genes involved in the regulation of sex determination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor. The wild-type fem-2 gene was cloned by identifying sequences from the C. elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals. cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues. Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele. This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C. elegans sex determination. Images PMID:8534913

  14. Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells.

    PubMed

    Kotake, K; Ozaki, N; Mizuta, M; Sekiya, S; Inagaki, N; Seino, S

    1997-11-21

    We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton. PMID:9367993

  15. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

    PubMed

    Scott, Emily E; Wolf, C Roland; Otyepka, Michal; Humphreys, Sara C; Reed, James R; Henderson, Colin J; McLaughlin, Lesley A; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P; Barnaba, Carlo; Brozik, James A; Jones, Jeffrey P; Estrada, D Fernando; Laurence, Jennifer S; Park, Ji Won; Backes, Wayne L

    2016-04-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  16. The cellular membrane as a mediator for small molecule interaction with membrane proteins.

    PubMed

    Mayne, Christopher G; Arcario, Mark J; Mahinthichaichan, Paween; Baylon, Javier L; Vermaas, Josh V; Navidpour, Latifeh; Wen, Po-Chao; Thangapandian, Sundarapandian; Tajkhorshid, Emad

    2016-10-01

    The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27163493

  17. Solid state NMR strategy for characterizing native membrane protein structures.

    PubMed

    Murray, Dylan T; Das, Nabanita; Cross, Timothy A

    2013-09-17

    Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment. Indeed, prior work has shown that this complex environment significantly influences transmembrane protein structure. Therefore, it is important to characterize such structures under conditions that closely resemble its native environment. Researchers have used two approaches to gain protein structural restraints via solid state NMR spectroscopy. The more traditional approach uses magic angle sample spinning to generate isotropic chemical shifts, much like solution NMR. As with solution NMR, researchers can analyze the backbone chemical shifts to obtain torsional restraints. They can also examine nuclear spin interactions between nearby atoms to obtain distances between atomic sites. Unfortunately, for membrane proteins in lipid preparations, the spectral resolution is not adequate to obtain complete resonance assignments. Researchers have developed another approach for gaining structural restraints from membrane proteins: the use of uniformly oriented lipid bilayers, which provides a method for obtaining high resolution orientational restraints. When the bilayers are aligned with respect to the magnetic field of the NMR spectrometer, researchers can obtain orientational restraints in which atomic sites in the protein are restrained relative to the alignment axis. However, this approach does not allow researchers to determine the relative packing between helices. By combining the two approaches, we can take advantage of the information acquired from each technique to minimize the challenges and maximize the quality of the

  18. Cloning of a putative juvenile hormone-responsive storage protein gene from the tobacco budworm, Heliothis virescens.

    PubMed

    Thompson, D M; Anspaugh, D D; Gahan, L J; Heckel, D G; Roe, R M

    1996-01-01

    A cDNA clone with 78% amino acid identity to a basic juvenile hormone (JH)-suppressible hemolymph protein from the cabbage looper, Trichoplusia ni, was isolated from the tobacco budworm, Heliothis virescens. This clone was obtained upon screening a cDNA library derived from larval fat body of a pesticide resistant strain of H. virescens with a cDNA probe for Drosophila melanogaster glutathione S-transferase. By comparison with other insect storage proteins, this clone was predicted to be part of an approximately 2,300 nucleotide (nt) cDNA, of which 691 nt were isolated and sequenced. The partial cDNA clone hybridizes to a RNA of approximately 2,370 nt in H. virescens. Treatment with a juvenoid (2-[1-methyl-2-(4-phenoxyphenoxy)ethoxy] pyridine; pyriproxifen) leads to a decrease in RNA levels of this putative hemolymph storage protein in early fifth stadium larvae of H. virescens, prior to commitment. In contrast, treatment in late fifth stadium (after commitment to pupal development) leads to an increase in the RNA level of this JH-responsive gene. This is the first report of both induction and suppression of storage protein RNA levels in the same stadium. We have given this gene the designation Hv-SP4 (H. virescens, storage protein 4; accession no. U48594). Genetic segregation analysis of restriction fragment length polymorphisms (RFLPs) defined by Hv-SP4 has shown that it is the product of a single-copy, Mendelian, autosomal gene. PMID:8756305

  19. Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes.

    PubMed Central

    Reusch, R N; Sadoff, H L

    1988-01-01

    A poly-beta-hydroxybutyrate complex extracted from the plasma membranes of genetically competent Escherichia coli contained polyhydroxybutyrate:polyphosphate:calcium in molar ratios approximating 1:1:0.5. The chain length of the polyhydroxybutyrate was estimated as 120-200 subunits, and that of the polyphosphate was estimated as 130-170 subunits. The extracted complex, when incorporated into liposomes, exhibited a lipid phase transition in the same temperature range as that of the membrane complex in whole cells as well as the same properties of irreversibility, lability, and sensitivity to chelating buffers. Space-filling molecular models and molecular energy minimization methods (Charmm) were used to develop and evaluate a plausible structure for the complex. It is proposed that the polyhydroxybutyrate forms an exolipophilic-endopolarophilic helix around an inner framework helix of calcium polyphosphate. The calcium ions link the two polymers by forming ionic bonds with phosphoryl oxygens of the polyphosphate and ion-dipole bonds with the ester carbonyl oxygens of the polyhydroxybutyrate. This symmetrical structure forms a channel through the membrane and may play a role in the transport of calcium, phosphate, and DNA. Images PMID:2454464

  20. Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

    PubMed Central

    Barros, Marilia; Jin, Danni; Lösche, Mathias; Vogt, Volker M.

    2015-01-01

    ABSTRACT The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with

  1. Three-Dimensional Crystals of Membrane Proteins: Bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Michel, Hartmut; Oesterhelt, Dieter

    1980-03-01

    The intrinsic membrane protein bacteriorhodopsin has been crystallized by salt precipitation after solubilization by octyl glucoside. Two different crystal forms were obtained, depending on the nature of the salt used and the pH. Needles formed in the presence of sodium phosphate and in ammonium sulfate solutions above pH 4.8. Cubes appeared in sodium citrate solutions or ammonium sulfate. Unlike the cubic crystals, the birefringent needles showed strong linear dichroism, which allowed determination of the orientation of the chromophore's transition moment. The procedure described here may be of general use in crystallographic studies of membrane proteins.

  2. Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms.

    PubMed

    Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

    2016-07-01

    This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26766517

  3. Polyisoprenylated methylated protein methyl esterase as a putative drug target for androgen-insensitive prostate cancer

    PubMed Central

    Poku, Rosemary A; Amissah, Felix; Duverna, Randolph; Aguilar, Byron J; Kiros, Gebre-Egziabher; Lamango, Nazarius S

    2014-01-01

    Prostate cancer (CaP) is the most frequently diagnosed cancer in US men, with an estimated 236,590 new cases and 29,720 deaths in 2013. There exists the need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant disease. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins have been implicated in CaP. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyses the only reversible and terminal reaction of the polyisoprenylation pathway and may promote the effects of G proteins on cell viability. In this review, the potential role of PMPMEase to serve as a new drug target for androgen-insensitive CaP was determined. Specific PMPMEase activities were found to be 3.5- and 4.5-fold higher in androgen-sensitive 22Rv1 and androgen-dependent LNCaP and 1.5- and 9.8-fold higher in castration-resistant DU 145 and PC-3 CaP cells compared to normal WPE1-NA22 prostate cells. The PMPMEase inhibitor, L-28, induced apoptosis with EC50 values ranging from 1.8 to 4.6 μM. The PMPMEase activity in the cells following treatment with L-28 followed a similar profile, with IC50 ranging from 2.3 to 130 μM. L-28 disrupted F-actin filament organisation at 5 μM and inhibited cell migration 4-fold at 2 μM. Analysis of a CaP tissue microarray for PMPMEase expression revealed intermediate, strong, and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to trace and weak staining in the normal and normal-adjacent tissue controls. The data are an indication that effective targeting of PMPMEase through the development of more potent agents may lead to the successful treatment of metastatic CaP. PMID:25228915

  4. New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

    PubMed Central

    Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

    2014-01-01

    Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

  5. Disulfide-bonded outer membrane proteins in the genus Legionella.

    PubMed Central

    Butler, C A; Street, E D; Hatch, T P; Hoffman, P S

    1985-01-01

    Legionella pneumophila and related species were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for outer membrane proteins. Of the 10 species examined, 9 contained a 24-kilodalton (kDa) major outer membrane protein (MOMP) that was resolvable only when outer membrane material was heated in the presence of 2-mercaptoethanol. Labeling studies with [35S]cysteine indicated that the protein contained cysteine, and disulfide cross-linking of the unreduced complex was demonstrated by labeling with iodoacetamide. The unreduced outer membrane preparation contained peptidoglycan, and after treatment with lysozyme to remove peptidoglycan, a protein complex of 95 kDa was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Reduction of the 95-kDa complex yielded 24-kDa monomers, suggesting that the 95-kDa complex was composed of four subunits. The 24-kDa MOMP from L. pneumophila was purified, and antibody produced to this protein cross-reacted with all species of Legionella as determined from an immunoblot of a sodium dodecyl sulfate gel. Only serogroup 1 strains of L. bozemanii lacked the 24-kDa MOMP and showed no cross-reactivity. These results suggest that the 24-kDa MOMP common to most species of Legionella contains a genus-specific epitope. Images PMID:3980079

  6. Purification of a membrane protein with conjugated engineered micelles.

    PubMed

    Patchornik, Guy; Danino, Dganit; Kesselman, Ellina; Wachtel, Ellen; Friedman, Noga; Sheves, Mordechai

    2013-07-17

    A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, β-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe(2+) ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 °C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins. PMID:23758098

  7. Detergent interaction with tethered bilayer lipid membranes for protein reconstitution

    NASA Astrophysics Data System (ADS)

    Broccio, Matteo; Zan Goh, Haw; Loesche, Mathias

    2009-03-01

    Tethered bilayer lipid membranes (tBLMs) are self-assembled biomimetic structures in which the membrane is separated from a solid substrate by a nm-thick hydrated submembrane space. These model systems are being used in binding studies of peripheral proteins and exotoxins. Here we aim at their application for the reconstitution of water-insoluble integral membrane proteins. As an alternative to fusion of preformed proteoliposomes we study the direct reconstitution of such proteins for applications in biosensing and pharmaceutical screening. For reconstitution, highly insulating tBLMs (R˜10^5-10^6 φ) were temporarily incubated with a detergent to screen for conditions that keep the detergent-saturated membranestable and ready to incorporate detergent-solubilized proteins. We assess the electrical characteristics, i.e. specific resistance and capacitance, by means of electrochemical impedance spectroscopy (EIS) under timed incubation with decylmaltoside and dodecylmaltoside detergents in a regime around their critical micelle concentration, 1.8 mM and 0.17 mM respectively and demonstrate the restoration of the tBLM upon detergent removal. Thereby a range of concentration and incubation times was identified, that represents optimal conditions for the subsequent membrane protein reconstitution.

  8. Erythrocyte membrane proteins in copper-deficient rats

    SciTech Connect

    Johnson, W.T.; Kramer, T.R.

    1987-05-01

    Increased osmotic stability and decreased survivability of erythrocytes caused by Cu deficiency suggest that low copper status may lead to modification in the organization of erythrocyte membrane proteins. Accordingly Cu deficiency was produced in rats by feeding a diet containing < 1 ppm Cu. The effects of low copper status on erythrocyte membrane proteins were assessed by sodium dodecyl sulfate polyacylamide electrophoresis. A 170,000 dalton protein (170K) amounted to 2.68 +/- 0.11% of the total membrane protein in erythrocytes from copper-deficient rats (n = 25) and 1.42 +/- 0.10% in erythrocytes from rats fed adequate Cu. When erythrocyte membranes from copper-deficient rats were extracted with 0.5% (v/v) Triton X-100, 170K remained associated with the cytoskeletal proteins, spectrin and actin. Thus, copper deficiency can alter the composition of the erythrocyte cytoskeleton. Furthermore, hematocrit levels in copper-deficient rats were negatively correlated to the amount of 170K suggesting that alteration of the erythrocyte cytoskeleton may be a factor that contributes to the anemia associated with copper deficiency.

  9. Tau protein binds to pericentromeric DNA: a putative role for nuclear tau in nucleolar organization.

    PubMed

    Sjöberg, Marcela K; Shestakova, Elena; Mansuroglu, Zeyni; Maccioni, Ricardo B; Bonnefoy, Eliette

    2006-05-15

    The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. A nuclear form of tau has been described in neuronal and non-neuronal cells, which displays a nucleolar localization during interphase but is associated with nucleolar-organizing regions in mitotic cells. In the present study, based on immunofluorescence, immuno-FISH and confocal microscopy, we show that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-rich alpha-satellite DNA sequences organized as constitutive heterochromatin. By using gel retardation, we demonstrate that tau not only colocalizes with, but also specifically binds to, AT-rich satellite DNA sequences apparently through the recognition of AT-rich DNA stretches. Here we propose a functional role for nuclear tau in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes. Since nuclear tau has also been found in neurons from patients with Alzheimer's disease (AD), aberrant nuclear tau could affect the nucleolar organization during the course of AD. We discuss nucleolar tau associated with AT-rich alpha-satellite DNA sequences as a potential molecular link between trisomy 21 and AD. PMID:16638814

  10. Role of OsHAL3 protein, a putative 4'-phosphopantothenoylcysteine decarboxylase in rice.

    PubMed

    Zhang, Ning; Wang, Xuechen; Chen, Jia

    2009-01-01

    In this study, we cloned the OsHAL3 gene from rice Oryza sativa. Alignment analysis revealed that OsHAL3 has a high sequence identity to Dfp protein in Escherichia coli and AtHAL3a protein in Arabidopsis thaliana, which have 4'-phosphopantothenoylcysteine decarboxylase (PPC-DC) activity. OsHAL3 can complement mutation in the E. coli dfp gene encoding PPC-DC, so that the mutant strains with OsHAL3 can grow on rich media at 42 degrees C and on VB minimal media at 30 degrees C. Complementation tests with point mutations of OsHAL3 suggested that the conserved Cys176 residue of OsHAL3 is a key active-site residue. The mutant OsHAL3 G180A has a partly reduced activity. Related mRNA-level analysis showed that the OsHAL3 gene is induced by calcium pantothenate in rice. PMID:19232050

  11. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  12. FUNCTIONALITY OF MEMBRANE SEPARATED EGG WHITE PROTEINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The excellent nutritional and functional properties of liquid egg white (LEW), which is essentially a viscous fat-free protein solution, are exploited in many food preparations. Thermal pasteurization (at 56.6oC for 3.5 min. minimum) is currently used by industry to eliminate the microflora in LEW ...

  13. Peripheral membrane proteins: Tying the knot between experiment and computation.

    PubMed

    Monje-Galvan, Viviana; Klauda, Jeffery B

    2016-07-01

    Experimental biology has contributed to answer questions about the morphology of a system and how molecules organize themselves to maintain a healthy functional cell. Single-molecule techniques, optical and magnetic experiments, and fluorescence microscopy have come a long way to probe structural and dynamical information at multiple scales. However, some details are simply too small or the processes are too short-lived to detect by experiments. Computational biology provides a bridge to understand experimental results at the molecular level, makes predictions that have not been seen in vivo, and motivates new fields of research. This review focuses on the advances on peripheral membrane proteins (PMPs) studies; what is known about their interaction with membranes, their role in cell biology, and some limitations that both experiment and computation still have to overcome to gain better structural and functional understanding of these PMPs. As many recent reviews have acknowledged, interdisciplinary efforts between experiment and computation are needed in order to have useful models that lead future directions in the study of PMPs. We present new results of a case study on a PMP that behaves as an intricate machine controlling lipid homeostasis between cellular organelles, Osh4 in yeast Saccharomyces cerevisiae. Molecular dynamics simulations were run to examine the interaction between the protein and membrane models that reflect the lipid diversity of the endoplasmic reticulum and trans-Golgi membranes. Our study is consistent with experimental data showing several residues that interact to smaller or larger extent with the bilayer upon stable binding (~200 ns into the trajectory). We identified PHE239 as a key residue stabilizing the protein-membrane interaction along with two other binding regions, the ALPS-like motif and the β6-β7 loops in the mouth region of the protein. This article is part of a Special Issue entitled: Membrane Proteins edited by J

  14. Inhibitors of Protein Translocation Across the ER Membrane.

    PubMed

    Kalies, Kai-Uwe; Römisch, Karin

    2015-10-01

    Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast-growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors. Inhibitors of protein transport across the ER membrane range from broad-spectrum to highly substrate-specific and can interfere with virtually any stage of this multistep process, and even with transport of endocytosed antigens into the cytosol for cross-presentation. PMID:26122014

  15. Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

    PubMed Central

    Hatahet, Feras; Blazyk, Jessica L.; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E.; Beckwith, Jonathan; Boyd, Dana

    2015-01-01

    Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants. PMID:26598701

  16. A Model for Shaping Membrane Sheets by Protein Scaffolds.

    PubMed

    Schweitzer, Yonatan; Shemesh, Tom; Kozlov, Michael M

    2015-08-01

    Membranes of peripheral endoplasmic reticulum form intricate morphologies consisting of tubules and sheets as basic elements. The physical mechanism of endoplasmic-reticulum shaping has been suggested to originate from the elastic behavior of the sheet edges formed by linear arrays of oligomeric protein scaffolds. The heart of this mechanism, lying in the relationships between the structure of the protein scaffolds and the effective intrinsic shapes and elastic properties of the sheets' edges, has remained hypothetical. Here we provide a detailed computational analysis of these issues. By minimizing the elastic energy of membrane bending, we determine the effects of a rowlike array of semicircular arclike membrane scaffolds on generation of a membrane fold, which shapes the entire membrane surface into a flat double-membrane sheet. We show, quantitatively, that the sheet's edge line tends to adopt a positive or negative curvature depending on the scaffold's geometrical parameters. We compute the effective elastic properties of the sheet edge and analyze the dependence of the equilibrium distance between the scaffolds along the edge line on the scaffold geometry. PMID:26244738

  17. Protein–protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins

    PubMed Central

    Kleanthous, Colin; Rassam, Patrice; Baumann, Christoph G

    2015-01-01

    It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises. PMID:26629934

  18. Putative sperm fusion protein IZUMO and the role of N-glycosylation

    SciTech Connect

    Inoue, Naokazu; Ikawa, Masahito; Okabe, Masaru

    2008-12-19

    IZUMO is the mouse sperm protein proven to be essential for fusion with eggs. It contains one immunoglobulin-like domain with a conserved glycosylation site within. In the present paper, we produced transgenic mouse lines expressing unglycosylated IZUMO (N204Q-IZUMO) in Izumo1 -/- background. The expression of N204Q-IZUMO rescued the infertile phenotype of IZUMO disrupted mice, indicating glycosylation is not essential for fusion-facilitating activity of IZUMO. The N204Q-IZUMO was produced in testis in comparable amounts to wild-type IZUMO, but the amount of N204Q-IZUMO on sperm was significantly decreased by the time sperm reached the cauda epididymis. These data suggest that glycosylation is not essential for the function of IZUMO, but has a role in protecting it from fragmentation in cauda epididymis.

  19. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain.

    PubMed

    Huala, E; Oeller, P W; Liscum, E; Han, I S; Larsen, E; Briggs, W R

    1997-12-19

    The NPH1 (nonphototropic hypocotyl 1) gene encodes an essential component acting very early in the signal-transduction chain for phototropism. Arabidopsis NPH1 contains a serine-threonine kinase domain and LOV1 and LOV2 repeats that share similarity (36 to 56 percent) with Halobacterium salinarium Bat, Azotobacter vinelandii NIFL, Neurospora crassa White Collar-1, Escherichia coli Aer, and the Eag family of potassium-channel proteins from Drosophila and mammals. Sequence similarity with a known (NIFL) and a suspected (Aer) flavoprotein suggests that NPH1 LOV1 and LOV2 may be flavin-binding domains that regulate kinase activity in response to blue light-induced redox changes. PMID:9405347

  20. Temperature-induced alteration of inositolphosphorylceramides in the putative glycosylated lipid precursors of Tetrahymena mimbres glycosylphosphatidylinositol-anchored proteins.

    PubMed Central

    Hung, C Y; Ko, Y G; Thompson, G A

    1995-01-01

    Tetrahymena species conta