Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

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Jaiswal Dinesh Kumar

2012-10-01

Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

Science.gov (United States)

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Mitofilin complexes: conserved organizers of mitochondrial membrane architecture.

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Zerbes, Ralf M; van der Klei, Ida J; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-11-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

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Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

2017-12-01

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

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Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

DEFF Research Database (Denmark)

Selao, Tiago Toscano; Branca, Rui; Chae, Pil Seok

2011-01-01

of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially...

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

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Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

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Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Mitofilin complexes : conserved organizers of mitochondrial membrane architecture

NARCIS (Netherlands)

Zerbes, Ralf M.; van der Klei, Ida J.; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-01-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner

Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

OpenAIRE

Quiroga, Rodrigo; Trenchi, Alejandra; Gonzalez Montoro, Ayelén; Valdez, Javier Esteban; Maccioni, Hugo Jose Fernando

2017-01-01

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the ...

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

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

2014-10-21

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

A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

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Sujoy Lahiri

2014-10-01

Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

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Lynn G.L. Richardson

2014-06-01

Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

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Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

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Boughton, Andrew P; Yang, Pei; Tesmer, Valerie M; Ding, Bei; Tesmer, John J G; Chen, Zhan

2011-09-13

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

Kinetics and Thermodynamics of Membrane Protein Folding

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Ernesto A. Roman

2014-03-01

Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.

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Yun-Shiang Chang

Full Text Available BACKGROUND: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus, is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. PRINCIPAL FINDINGS: In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. CONCLUSIONS: From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

Mitochondrial cardiolipin/phospholipid trafficking: the role of membrane contact site complexes and lipid transfer proteins.

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Schlattner, Uwe; Tokarska-Schlattner, Malgorzata; Rousseau, Denis; Boissan, Mathieu; Mannella, Carmen; Epand, Richard; Lacombe, Marie-Lise

2014-04-01

Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

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Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Isomeric Detergent Comparison for Membrane Protein Stability

DEFF Research Database (Denmark)

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

2016-01-01

and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

Energy Technology Data Exchange (ETDEWEB)

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

2004-12-17

Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

Membrane Tethering Complexes in the Endosomal System

OpenAIRE

Spang, Anne

2016-01-01

Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the class C core vacuole/endosome tethering (CORVET) complex, while fusion of late endosomes with lysosomes depends on the homotypic fusion and vacuole protein sorting (HOPS) complex. Recycling through the trans-Golgi network (TGN) and to the plasma membrane is...

Membrane tethering complexes in the endosomal system

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Anne eSpang

2016-05-01

Full Text Available Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the CORVET complex, while fusion of late endosomes with lysosomes depends on the HOPS complex. Recycling through the TGN and to the plasma membrane is facilitated by the GARP and EARP complexes, respectively. However, there are other tethering functions in the endosomal system as there are multiple pathways through which proteins can be delivered from endosomes to either the TGN or the plasma membrane. Furthermore, complexes that may be part of novel tethering complexes have been recently identified. Thus it is likely that more tethering factors exist. In this review, I will provide an overview of different tethering complexes of the endosomal system and discuss how they may provide specificity in membrane traffic.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

Science.gov (United States)

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

NARCIS (Netherlands)

Peden, A.A.; Oorschot, V.; Hesser, B.A.; Austin, C.D.; Scheller, R.H.; Klumperman, J.

2004-01-01

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal

Electron microscopy of cyanobacterial membrane proteins

NARCIS (Netherlands)

Folea, Ioana Mihaela

2008-01-01

The main focus of this thesis is photosynthetic protein complexes, and their organization within the membrane of cyanobacteria. In cyanobacteria large proteins catalyze the light reactions of photosynthesis. One of the key proteins is photosystem II. We have found for the first time by electron

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris.

Science.gov (United States)

Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min

2017-10-01

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l -1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of UV-irradiation on DNA-membrane complex of Bacillus subtilis

International Nuclear Information System (INIS)

Chefranova, O.A.; Gaziev, A.I.

1979-01-01

The UV radiation effect on DNA membrane complex of Bacillus subtilis has been studied. Increase of DNA content in the DNA membrane complex in two strains of 168 and recA - and its decrease in the polA - strain are shown. The above effect in the first two stamms is suppressed with caffeine and correlates with the change in protein content in the DNA membrane complex, determined by a radioactive label, but not lipids in other words, fixation of DNA and membrane goes through proteins. Capability of DNA content increase in the DNA membrane complex after UV irradiation and subsequent bacteria incubation in a total medium correlates with the relative sensitivity of stamm UV sensitivity. It is suggested, that the reparation synthesis goes in cells on the membrane and that binding of DNA and the membrane is necessary for the normal DNA reparation process

Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron

International Nuclear Information System (INIS)

Xu, Qingping; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2010-01-01

The crystal structure of a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed. Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT-3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment

Complex Dynamic Development of Poliovirus Membranous Replication Complexes

Science.gov (United States)

Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

2012-01-01

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

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

Science.gov (United States)

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.

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

Directory of Open Access Journals (Sweden)

Li Weijun

2011-01-01

Full Text Available Abstract Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins, which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin.

Science.gov (United States)

Zheng, Jianhua; Wei, Candong; Zhao, Lina; Liu, Liguo; Leng, Wenchuan; Li, Weijun; Jin, Qi

2011-01-18

Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. In this

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

Science.gov (United States)

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

Science.gov (United States)

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A tethering complex drives the terminal stage of SNARE-dependent membrane fusion

Science.gov (United States)

D'Agostino, Massimo; Risselada, Herre Jelger; Lürick, Anna; Ungermann, Christian; Mayer, Andreas

2017-11-01

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

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

Energy Technology Data Exchange (ETDEWEB)

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

The role of antioxidant-protein interactions in biological membrane

International Nuclear Information System (INIS)

McGillivray, Duncan J; Singh, Rachna; Melton, Laurence D.; Worcester, David L.; Gilbert, Elliot P.

2009-01-01

Full text: Oxidative damage of cellular membranes has been linked to a variety of disease pathologies, including cardiac disease, Alzheimer's and complications due to diabetes. The oxidation of unsaturated and polyunsaturated fatty acid chains found in cellular membranes leads to significant alteration in membrane physical properties, including lipid orientation and membrane permeability, which ultimately affect biological function. Polyphenols are naturally occurring phytochemicals present in a number of fruit and vegetables that are of interest for their anti-oxidative powers. These polyphenols inhibit lipid oxidation in cellular membrane surfaces, although the mechanism of this inhibition is not entirely clear. Moreover, the polyphenols have significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein-polyphenol complexes Significantly, in the presence of casein proteins the oxidation inhibition the polyphenols in the membrane is significantly enhanced (as assessed by Lipid Peroxidation Inhibition Capacity assays). Thus the antioxidant pathway appears to involve these protein/polyphenol complexes, as well as direct antioxidant action by the polyphenol. Here we discuss neutron and x-ray scattering results from phospholipid membranes, looking at the positioning of two examples of polyphenolic antioxidants in phospholipid membranes, quercetin and phloretin, the antioxidants' impact on the membrane organisation, and the interaction between antioxidant and extra-membranous protein. This information sheds light on the mechanism of antioxidant protection in these systems, which may be used to understand biological responses to oxidative stress.

Native proteomic analysis of protein complexes in murine intestinal brush border membranes

Czech Academy of Sciences Publication Activity Database

Babušiak, M.; Man, Petr; Petrák, J.; Vyoral, D.

2007-01-01

Roč. 7, č. 1 (2007), s. 121-129 ISSN 1615-9853 R&D Projects: GA ČR(CZ) GD204/03/H066; GA AV ČR KJB500200612; GA MŠk LC545 Grant - others:GA ČR(CZ) GA303/04/0003; GA MZd(CZ) NR8930; GA MŠk(CZ) LC06044; CZ(CZ) 023736; GA MZd(CZ) NR8317 Program:NR Institutional research plan: CEZ:AV0Z50200510 Keywords : blue native electrophoresis * brush border membranes * protein complexes Subject RIV: EE - Microbiology, Virology Impact factor: 5.479, year: 2007

Synchrotron small-angle x-ray scattering investigation on integral membrane protein light-harvesting complex LH2 from photosynthetic bacterium rhodopseudomonas acidophila

International Nuclear Information System (INIS)

Du Luchao; Weng Yuxiang; Hong Xinguo; Xian Dingchang; Kobayashi Katsumi

2006-01-01

Structures of membrane protein in solution are different from that in crystal phase. We present the primary results of small angle x-ray scattering (SAXS) resolved topological structures of a light harvesting antenna membrane protein complex LH2 from photosynthetic bacteria Rhodopseudomonas acidophila in detergent solution for the first time. Our results show that the elliptical shape of the LH2 complex in solution clearly deviates from its circular structure in crystal phase determined by x-ray diffraction. This result provides an insight into the structure and function interplay in LH2. (authors)

Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

DEFF Research Database (Denmark)

Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

2017-01-01

of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

Structural Aspects of Bacterial Outer Membrane Protein Assembly.

Science.gov (United States)

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.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

Science.gov (United States)

Rumak, Izabela; Mazur, Radosław; Gieczewska, Katarzyna; Kozioł-Lipińska, Joanna; Kierdaszuk, Borys; Michalski, Wojtek P; Shiell, Brian J; Venema, Jan Henk; Vredenberg, Wim J; Mostowska, Agnieszka; Garstka, Maciej

2012-05-25

The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

Science.gov (United States)

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

International Nuclear Information System (INIS)

Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

1976-01-01

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

Protein diffusion in plant cell plasma membranes: the cell-wall corral.

Science.gov (United States)

Martinière, Alexandre; Runions, John

2013-01-01

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Assembly factors for the membrane arm of human complex I.

Science.gov (United States)

Andrews, Byron; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2013-11-19

Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron-sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.

Identification of membrane proteins by tandem mass spectrometry of protein ions

Science.gov (United States)

Carroll, Joe; Altman, Matthew C.; Fearnley, Ian M.; Walker, John E.

2007-01-01

The most common way of identifying proteins in proteomic analyses is to use short segments of sequence (“tags”) determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning α-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1–4 transmembrane α-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5–18 transmembrane α-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase. PMID:17720804

The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

NARCIS (Netherlands)

Huisman, I.H.; Prádanos, P.; Hernández, A.

2000-01-01

It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

An Integrated Framework Advancing Membrane Protein Modeling and Design.

Directory of Open Access Journals (Sweden)

Rebecca F Alford

2015-09-01

Full Text Available 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.

Protein diffusion in plant cell plasma membranes: The cell-wall corral

Directory of Open Access Journals (Sweden)

Alexandre eMartinière

2013-12-01

Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study

DEFF Research Database (Denmark)

Das, Manabendra; Du, Yang; Ribeiro, Orquidea

2017-01-01

were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes......Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties....... Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n...

Exploring the Spatiotemporal Organization of Membrane Proteins in Living Plant Cells.

Science.gov (United States)

Wang, Li; Xue, Yiqun; Xing, Jingjing; Song, Kai; Lin, Jinxing

2018-04-29

Plasma membrane proteins have important roles in transport and signal transduction. Deciphering the spatiotemporal organization of these proteins provides crucial information for elucidating the links between the behaviors of different molecules. However, monitoring membrane proteins without disrupting their membrane environment remains difficult. Over the past decade, many studies have developed single-molecule techniques, opening avenues for probing the stoichiometry and interactions of membrane proteins in their native environment by providing nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we assess recent progress in the development of labeling and imaging technology for membrane protein analysis. We focus in particular on several single-molecule techniques for quantifying the dynamics and assembly of membrane proteins. Finally, we provide examples of how these new techniques are advancing our understanding of the complex biological functions of membrane proteins.

The study of membrane-protein /detergent interactions by neutron crystallography

Energy Technology Data Exchange (ETDEWEB)

Timmins, P A; Penel, S [Institut Max von Laue - Paul Langevin (ILL), 38 - Grenoble (France); Pebay-Peyroula, E [IBS- UJF Grenoble (France)

1997-04-01

Proteins which are found embedded in membranes can usually only be purified and studied from the point of view of structure by dissolving them in detergents. The structure of the resulting mixed protein-detergent complexes are poorly understood. An important method for studying them is through neutron diffraction of the crystalline complexes. This allows us to understand better how the proteins behave in the natural membrane as well as allowing us to visualize and hopefully improve the crystallisation process. Studies on the pore-forming protein porin using data collected on the diffractometer DB21 are described. (author). 4 refs.

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

Directory of Open Access Journals (Sweden)

Choe Senyon

2007-11-01

Full Text Available Abstract Background Structural studies of integral membrane proteins (IMPs are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs. The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening. Conclusion The techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

Science.gov (United States)

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

Science.gov (United States)

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

NARCIS (Netherlands)

Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

2010-01-01

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

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N. G. Zemlianskykh

2016-10-01

Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

Science.gov (United States)

Vasiljev, Andreja; Ahting, Uwe; Nargang, Frank E; Go, Nancy E; Habib, Shukry J; Kozany, Christian; Panneels, Valérie; Sinning, Irmgard; Prokisch, Holger; Neupert, Walter; Nussberger, Stephan; Rapaport, Doron

2004-03-01

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Membranes and mammalian glycolipid transferring proteins.

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Tuuf, Jessica; Mattjus, Peter

2014-02-01

Glycolipids are synthesized in and on various organelles throughout the cell. Their trafficking inside the cell is complex and involves both vesicular and protein-mediated machineries. Most important for the bulk lipid transport is the vesicular system, however, lipids moved by transfer proteins are also becoming more characterized. Here we review the latest advances in the glycolipid transfer protein (GLTP) and the phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) field, from a membrane point of view. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins

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Carroll, Joe; Fearnley, Ian M.; Walker, John E.

2006-01-01

The covalent structure of a protein is incompletely defined by its gene sequence, and mass spectrometric analysis of the intact protein is needed to detect the presence of any posttranslational modifications. Because most membrane proteins are purified in detergents that are incompatible with mass spectrometric ionization techniques, this essential measurement has not been made on many hydrophobic proteins, and so proteomic data are incomplete. We have extracted membrane proteins from bovine mitochondria and detergent-purified NADH:ubiquinone oxidoreductase (complex I) with organic solvents, fractionated the mixtures by hydrophilic interaction chromatography, and measured the molecular masses of the intact membrane proteins, including those of six subunits of complex I that are encoded in mitochondrial DNA. These measurements resolve long-standing uncertainties about the interpretation of the mitochondrial genome, and they contribute significantly to the definition of the covalent composition of complex I. PMID:17060615

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

Science.gov (United States)

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

The TIC complex uncovered: The alternative view on the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts.

Science.gov (United States)

Nakai, Masato

2015-09-01

Chloroplasts must import thousands of nuclear-encoded preproteins synthesized in the cytosol through two successive protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to fulfill their complex physiological roles. The molecular identity of the TIC translocon had long remained controversial; two proteins, namely Tic20 and Tic110, had been proposed to be central to protein translocation across the inner envelope membrane. Tic40 also had long been considered to be another central player in this process. However, recently, a novel 1-megadalton complex consisting of Tic20, Tic56, Tic100, and Tic214 was identified at the chloroplast inner membrane of Arabidopsis and was demonstrated to constitute a general TIC translocon which functions in concert with the well-characterized TOC translocon. On the other hand, direct interaction between this novel TIC transport system and Tic110 or Tic40 was hardly observed. Consequently, the molecular model for protein translocation across the inner envelope membrane of chloroplasts might need to be extensively revised. In this review article, I intend to propose such alternative view regarding the TIC transport system in contradistinction to the classical view. I also would emphasize importance of reevaluation of previous works in terms of with what methods these classical Tic proteins such as Tic110 or Tic40 were picked up as TIC constituents at the very beginning as well as what actual evidence there were to support their direct and specific involvement in chloroplast protein import. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

Science.gov (United States)

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

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Sébastien Besteiro

2009-02-01

Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

Channel crossing: how are proteins shipped across the bacterial plasma membrane?

Science.gov (United States)

Collinson, Ian; Corey, Robin A; Allen, William J

2015-10-05

The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation--the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins. © 2015 The Authors.

Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p

Energy Technology Data Exchange (ETDEWEB)

Josyula, Ratnakar [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Jin, Zhongmin [SER-CAT, APS, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 (United States); McCombs, Deborah; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

2006-02-01

Tim44p is an essential mitochondrial peripheral membrane protein. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6{sub 3}22, with unit-cell parameters a = 124.25, c = 77.83 Å. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.

Nanodisc-solubilized membrane protein library reflects the membrane proteome

OpenAIRE

Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

2013-01-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

Science.gov (United States)

Dubey, Rashmi; Harrison, Brooke; Dangoudoubiyam, Sriveny; Bandini, Giulia; Cheng, Katherine; Kosber, Aziz; Agop-Nersesian, Carolina; Howe, Daniel K; Samuelson, John; Ferguson, David J P; Gubbels, Marc-Jan

2017-01-01

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis , IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma . IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin

SURVEY REGARDING THE ULTRAFILTRATION OF PROTEINES THROUGH MEMBRANE BASED PROCEDURES

Directory of Open Access Journals (Sweden)

CAMELIA HODOSAN

2008-05-01

Full Text Available This work is based on examples that emphasize the complexity of the proteins ultrafiltration process, pointing out the first 10-15 minutes of ultrafiltration. The knowledgement of the factors that influence the separation through ultrafiltration of proteins will allow to choose the right type of membrane, the frequent use of the same membrane and the operation in mechanical and chemical conditions adequate to the ultrafiltration system, when it is separated a protein with certain molecular weight.

Function of membrane protein in silica nanopores: incorporation of photosynthetic light-harvesting protein LH2 into FSM.

Science.gov (United States)

Oda, Ippei; Hirata, Kotaro; Watanabe, Syoko; Shibata, Yutaka; Kajino, Tsutomu; Fukushima, Yoshiaki; Iwai, Satoshi; Itoh, Shigeru

2006-01-26

A high amount of functional membrane protein complex was introduced into a folded-sheet silica mesoporous material (FSM) that has nanometer-size pores of honeycomb-like hexagonal cylindrical structure inside. The photosynthetic light-harvesting complex LH2, which is a typical membrane protein, has a cylindrical structure of 7.3 nm diameter and contains 27 bacteriochlorophyll a and nine carotenoid molecules. The complex captures light energy in the anoxygenic thermophilic purple photosynthetic bacterium Thermochromatium tepidum. The amount of LH2 adsorbed to FSM was determined optically and by the adsorption isotherms of N2. The FSM compounds with internal pore diameters of 7.9 and 2.7 nm adsorbed LH2 at 1.11 and 0.24 mg/mg FSM, respectively, suggesting the high specific affinity of LH2 to the interior of the hydrophobic nanopores with a diameter of 7.9 nm. The LH2 adsorbed to FSM showed almost intact absorption bands of bacteriochlorophylls, and was fully active in the capture and transfer of excitation energy. The LH2 complex inside the FSM showed increased heat stability of the exciton-type absorption band of bacteriochlorophylls (B850), suggesting higher circular symmetry. The environment inside the hydrophobic silica nanopores can be a new matrix for the membrane proteins to reveal their functions. The silica-membrane protein adduct will be useful for the construction of new probes and reaction systems.

Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

Energy Technology Data Exchange (ETDEWEB)

Lelievre, Sophie A; Bissell, Mina J

1998-10-21

Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

Science.gov (United States)

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

Science.gov (United States)

Golan, Yonatan; Sherman, Eilon

2017-06-01

The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

Analysis of proteins in Chlamydia trachomatis L2 outer membrane complex, COMC

DEFF Research Database (Denmark)

Birkelund, Svend; Morgan-Fisher, Marie; Timmerman, Evy

2009-01-01

The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary a...

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Science.gov (United States)

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Analytical use of multi-protein Fluorescence Resonance Energy Transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes.

Science.gov (United States)

Krause, Christopher D; Izotova, Lara S; Pestka, Sidney

2013-10-01

Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-γR1, IL-10R1 and IFN-λR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-λR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-γR1 and IFN-γR2 or between IFN-αR1 and IFN-αR2c increases, FRET among other pairs of proteins changes within each complex. Copyright © 2013 Elsevier Ltd. All rights reserved.

Membrane proteins bind lipids selectively to modulate their structure and function.

Science.gov (United States)

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

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

Directory of Open Access Journals (Sweden)

Helene eSanfacon

2013-01-01

Full Text Available 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 trans-membrane 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.

Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone

International Nuclear Information System (INIS)

Ozegovic, B.; Dobrovic-Jenik, D.; Milkovic, S.

1988-01-01

The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3 H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3 H-aldosterone - kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3 H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3 H-aldosterone peak while the second 3 H-aldosterone peak was eluted with Ve corresponding to Ve of free 3 H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3 H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass. (author)

Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

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Benjamin Clémençon

2012-02-01

Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

Lipid nanotechnologies for structural studies of membrane-associated proteins.

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Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

2014-11-01

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

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Bartosz Różycki

Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

Peripheral Protein Unfolding Drives Membrane Bending.

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Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

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Emilie eGauliard

2015-02-01

Full Text Available Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins. Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

International Nuclear Information System (INIS)

Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

1981-01-01

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins

Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

NARCIS (Netherlands)

Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

1996-01-01

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

International Nuclear Information System (INIS)

Kangur, L; Leiger, K; Freiberg, A

2008-01-01

The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes

Evidence for high-pressure-induced rupture of hydrogen bonds in LH2 photosynthetic antenna pigment-protein complexes

Energy Technology Data Exchange (ETDEWEB)

Kangur, L; Leiger, K; Freiberg, A [Institute of Physics, University of Tartu, Riia 142, Tartu 51014 (Estonia)

2008-07-15

The bacteriochlorophyll a-containing LH2 light harvesting complex is an integral membrane protein that catalyzes the photosynthetic process in purple photosynthetic bacteria. The LH2 complexes from Rhodobacter sphaeroides show characteristic strong absorbance at 800 and 850 nm due to the bacteriochlorophyll a molecules confined in two separate areas of the protein. Using these cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high hydrostatic pressure up to 2.1 GPa of LH2 complexes embedded into natural membrane environment or extracted with detergent. We demonstrate that high pressure does induce significant alterations to the tertiary structure of the protein in proximity of the protein-bound bacteriochlorophyll a molecules, including breakage of the hydrogen bond they are involved in. The membrane-embedded complexes appear more resilient to damaging effects of the compression than the complexes extracted into detergent environment. This difference has tentatively been explained by more compact structure of the membrane-embedded complexes.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Science.gov (United States)

Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

2015-01-01

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

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Zhang Jian-Hua

2007-09-01

Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Science.gov (United States)

De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

2015-07-17

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

Science.gov (United States)

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Intracellular and transcellular transport of secretory and membrane proteins in the rat hepatocyte

International Nuclear Information System (INIS)

Sztul, E.S.

1984-01-01

The intra- and transcellular transport of hepatic secretory and membrane proteins was studied in rats in vivo using [ 3 H]fucose and [ 35 S]cyteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile and plasma were separated by SDS-PAGE and identified by fluorography. 3 H-radioactivity in Golgi fractions peaked at 10 min post injection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from the Golgi complex occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 10 min later than the bulk of content proteins. A major 80K form of Secretory Component (SC) was identified in the bile by precipitation with an anti IgA antibody. A comparative study of kinetics of transport of 35 S-labeled SC and 35 S-labeled albumin showed that albumin peaked in bile at ∼45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins which are delivered to the bile canaliculus (BC)

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

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O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

Membrane shape modulates transmembrane protein distribution.

Science.gov (United States)

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

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Interaction between La(III) and proteins on the plasma membrane of horseradish

Science.gov (United States)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

International Nuclear Information System (INIS)

Nielsen, Anders Lade

2009-01-01

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Energy Technology Data Exchange (ETDEWEB)

Nielsen, Anders Lade, E-mail: aln@humgen.au.dk [Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C (Denmark)

2009-10-23

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

Energy Technology Data Exchange (ETDEWEB)

Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

2011-06-30

Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer-membrane

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

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Marc Lenoir

2015-10-01

Full Text Available 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.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

Science.gov (United States)

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

2015-10-23

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.

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

Science.gov (United States)

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

2007-08-17

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

Complex interplay between the P-glycoprotein multidrug efflux pump and the membrane: its role in modulating protein function

Directory of Open Access Journals (Sweden)

Frances Jane Sharom

2014-03-01

Full Text Available Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1, which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly-directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behaviour of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Science.gov (United States)

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

Protein shape and crowding drive domain formation and curvature in biological membranes

NARCIS (Netherlands)

Frese, R.N.; Pamies, Josep C.; Olsen, John D.; Bahatyrova, S.; van der Weij-de Wit, Chantal D.; Aartsma, Thijs J.; Otto, Cornelis; Hunter, C. Neil; Frenkel, Daan; van Grondelle, Rienk

2007-01-01

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization

Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes: Implications for Lipid-Linked Disorders

OpenAIRE

Bogdanov, Mikhail; Mileykovskaya, Eugenia; Dowhan, William

2008-01-01

Lipids play important roles in cellular dysfunction leading to disease. Although a major role for phospholipids is in defining the membrane permeability barrier, phospholipids play a central role in a diverse range of cellular processes and therefore are important factors in cellular dysfunction and disease. This review is focused on the role of phospholipids in normal assembly and organization of the membrane proteins, multimeric protein complexes, and higher order supercomplexes. Since lipi...

The Origin and Early Evolution of Membrane Proteins

Science.gov (United States)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

Science.gov (United States)

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

Science.gov (United States)

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

Science.gov (United States)

Chevalier, Adrien S; Chaumont, François

2015-05-01

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Proteomic screen for multiprotein complexes in synaptic plasma membrane from rat hippocampus by blue native gel electrophoresis and tandem mass spectrometry.

Science.gov (United States)

Li, Xuanwen; Xie, Chunliang; Jin, Qihui; Liu, Mingjun; He, Quanyuan; Cao, Rui; Lin, Yong; Li, Jianglin; Li, Yan; Chen, Ping; Liang, Songping

2009-07-01

Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

Science.gov (United States)

Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

2014-01-01

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

Science.gov (United States)

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino

Biomimetic devices functionalized by membrane channel proteins

Science.gov (United States)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Proteins and Peptides in Biomimetic Polymeric Membranes

DEFF Research Database (Denmark)

Perez, Alfredo Gonzalez

2013-01-01

This chapter discusses recent advances and the main advantages of block copolymers for functional membrane protein reconstitution in biomimetic polymeric membranes. A rational approach to the reconstitution of membrane proteins in a functional form can be addressed by a more holistic view by using...... other kind of nonbiological amphiphilic molecules. An interesting possibility could be the use of self-assembled proteins in a lipid-free membrane mimicking the capside of some viruses. The membrane proteins that have been more actively used in combination with block copolymer membranes are gramicidin...

High yield cell-free production of integral membrane proteins without refolding or detergents.

Science.gov (United States)

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

Science.gov (United States)

Sahu, Indra D; Zhang, Rongfu; Dunagan, Megan M; Craig, Andrew F; Lorigan, Gary A

2017-06-01

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (T m ) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the T m titration experiment indicated an increase in T m values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

Science.gov (United States)

Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

2014-01-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins.

Science.gov (United States)

Ramakrishnan, N; Sunil Kumar, P B; Radhakrishnan, Ravi

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this

Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

Science.gov (United States)

Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

2014-01-01

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

DEFF Research Database (Denmark)

Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

2006-01-01

A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

Science.gov (United States)

Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

2016-01-01

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

Science.gov (United States)

Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

2014-09-03

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

[Interaction of FABP4 with plasma membrane proteins of endothelial cells].

Science.gov (United States)

Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

2015-01-01

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

Energy Technology Data Exchange (ETDEWEB)

Ramakrishnan, N., E-mail: ramn@seas.upenn.edu [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Bioengineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA-19104 (United States); Sunil Kumar, P.B., E-mail: sunil@physics.iitm.ac.in [Department of Physics, Indian Institute of Technology Madras, Chennai, 600036 (India); Radhakrishnan, Ravi, E-mail: rradhak@seas.upenn.edu [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Bioengineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA-19104 (United States)

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein–lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham–Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

International Nuclear Information System (INIS)

Ramakrishnan, N.; Sunil Kumar, P.B.; Radhakrishnan, Ravi

2014-01-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein–lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham–Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

International Nuclear Information System (INIS)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph; Bernatchez, Pascal

2011-01-01

Highlights: ► Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. ► Dysferlin interacts with key signaling proteins for transcytosis in EC. ► Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Energy Technology Data Exchange (ETDEWEB)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada); Bernatchez, Pascal, E-mail: pbernatc@mail.ubc.ca [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada)

2011-11-18

Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

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Kris E Spaeth

2009-09-01

Full Text Available In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone. Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion". The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

Modelling of proteins in membranes

DEFF Research Database (Denmark)

Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

2006-01-01

This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly ...

The absence of chlorophyll b affects lateral mobility of photosynthetic complexes and lipids in grana membranes of Arabidopsis and barley chlorina mutants.

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Tyutereva, Elena V; Evkaikina, Anastasiia I; Ivanova, Alexandra N; Voitsekhovskaja, Olga V

2017-09-01

The lateral mobility of integral components of thylakoid membranes, such as plastoquinone, xanthophylls, and pigment-protein complexes, is critical for the maintenance of efficient light harvesting, high rates of linear electron transport, and successful repair of damaged photosystem II (PSII). The packaging of the photosynthetic pigment-protein complexes in the membrane depends on their size and stereometric parameters which in turn depend on the composition of the complexes. Chlorophyll b (Chlb) is an important regulator of antenna size and composition. In this study, the lateral mobility (the mobile fraction size) of pigment-protein complexes and lipids in grana membranes was analyzed in chlorina mutants of Arabidopsis and barley lacking Chlb. In the Arabidopsis ch1-3 mutant, diffusion of membrane lipids decreased as compared to wild-type plants, but the diffusion of photosynthetic complexes was not affected. In the barley chlorina f2 3613 mutant, the diffusion of pigment-protein complexes significantly decreased, while the diffusion of lipids increased, as compared to wild-type plants. We propose that the size of the mobile fractions of pigment-protein complexes in grana membranes in vivo is higher than reported previously. The data are discussed in the context of the protein composition of antennae, characteristics of the plastoquinone pool, and production of reactive oxygen species in leaves of chlorina mutants.

Novel Tripod Amphiphiles for Membrane Protein Analysis

DEFF Research Database (Denmark)

Chae, Pil Seok; Kruse, Andrew C; Gotfryd, Kamil

2013-01-01

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution...

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

Science.gov (United States)

Jouhet, Juliette; Gray, John C

2009-10-01

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

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

Energy Technology Data Exchange (ETDEWEB)

Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

2015-06-15

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.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

Cationic membranes complexed with oppositely charged microtubules: hierarchical self-assembly leading to bio-nanotubes

International Nuclear Information System (INIS)

Raviv, Uri; Needleman, Daniel J; Safinya, Cyrus R

2006-01-01

The self-assembly of microtubules and charged membranes has been studied, using x-ray diffraction and electron microscopy. Polyelectrolyte lipid complexes usually form structures templated by the lipid phase, when the polyelectrolyte curvature is much larger than the membrane spontaneous curvature. When the polyelectrolyte curvature approaches the membrane spontaneous curvature, as in microtubules, two types of new structures emerge. Depending on the conditions, vesicles either adsorb onto the microtubule, forming a 'beads on a rod' structure, or coat the microtubule, which now forms the template. Tubulin oligomers then coat the external lipid layer, forming a lipid protein nanotube. The tubulin oligomer coverage at the external layer is determined by the membrane charge density. The energy barrier between the beads on a rod and the lipid-protein nanotube states depends on the membrane bending rigidity and membrane charge density. By controlling the lipid/tubulin stoichiometry we can switch between lipid-protein nanotubes with open ends to lipid-protein nanotubes with closed end with lipid cups. This forms the basis for controlled drug encapsulation and release

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

Directory of Open Access Journals (Sweden)

Deirdre R Ducken

Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

Controlling the shape of membrane protein polyhedra

Science.gov (United States)

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

2017-03-01

Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

Science.gov (United States)

Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

International Nuclear Information System (INIS)

Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf; Schrader, Michael

2010-01-01

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

Probing protein-lipid interactions by FRET between membrane fluorophores

Science.gov (United States)

Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

2016-09-01

Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

Xanthophylls as modulators of membrane protein function.

Science.gov (United States)

Ruban, Alexander V; Johnson, Matthew P

2010-12-01

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

Cytoskeletal Components Define Protein Location to Membrane Microdomains*

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Szymanski, Witold G.; Zauber, Henrik; Erban, Alexander; Gorka, Michal; Wu, Xu Na; Schulze, Waltraud X.

2015-01-01

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. PMID:26091700

Characterization of the human GARP (Golgi associated retrograde protein) complex

International Nuclear Information System (INIS)

Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank

2005-01-01

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

Science.gov (United States)

Pešić, Ivana; Müller, Gerhard A; Baumann, Cosima; Dihazi, Gry H; Koziolek, Michael J; Eltoweissy, Marwa; Bramlage, Carsten; Asif, Abdul R; Dihazi, Hassan

2013-04-01

The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of membrane association of Rinderpest virus matrix protein

International Nuclear Information System (INIS)

Subhashri, R.; Shaila, M.S.

2007-01-01

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

Current strategies for protein production and purification enabling membrane protein structural biology.

Science.gov (United States)

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions

International Nuclear Information System (INIS)

Szajner, Patricia; Jaffe, Howard; Weisberg, Andrea S.; Moss, Bernard

2004-01-01

Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature

Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

Directory of Open Access Journals (Sweden)

Heidi Koldsø

2014-10-01

Full Text Available Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2, in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

KAUST Repository

Ooi, Amanda Siok Lee

2016-07-19

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

KAUST Repository

Ooi, Amanda Siok Lee; Wong, Aloysius Tze; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Christoph A

2016-01-01

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

Production of membrane proteins without cells or detergents.

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Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes.

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Freiberg, Arvi; Rätsep, Margus; Timpmann, Kõu

2012-08-01

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. Copyright © 2011. Published by Elsevier B.V.

Modelling small-angle scattering data from complex protein-lipid systems

DEFF Research Database (Denmark)

Kynde, Søren Andreas Røssell

This thesis consists of two parts. The rst part is divided into five chapters. Chapter 1 gives a general introduction to the bio-molecular systems that have been studied. These are membrane proteins and their lipid environments in the form of phospholipid nanodiscs. Membrane proteins...... the techniques very well suited for the study of the nanodisc system. Chapter 3 explains two different modelling approaches that can be used in the analysis of small-angle scattering data from lipid-protein complexes. These are the continuous approach where the system of interest is modelled as a few regular...... combine the bene ts of each of the methods and give unique structural information about relevant bio-molecular complexes in solution. Chapter 4 describes the work behind a proposal of a small-angle neutron scattering instrument for the European Spallation Source under construction in Lund. The instrument...

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

Science.gov (United States)

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity.

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Antoine de Morrée

Full Text Available Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

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Pohorille, Andrew

2003-01-01

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4

Cell-free expression and stable isotope labelling strategies for membrane proteins

International Nuclear Information System (INIS)

Sobhanifar, Solmaz; Reckel, Sina; Junge, Friederike; Schwarz, Daniel; Kai, Lei; Karbyshev, Mikhail; Loehr, Frank; Bernhard, Frank; Doetsch, Volker

2010-01-01

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on α-helical transmembrane proteins which benefit most from such methods.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

Science.gov (United States)

Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

DEFF Research Database (Denmark)

Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

2007-01-01

Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae...... was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Science.gov (United States)

Zhang, Rongfu; Sahu, Indra D; Liu, Lishan; Osatuke, Anna; Comer, Raven G; Dabney-Smith, Carole; Lorigan, Gary A

2015-01-01

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment

Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling

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Tzviya Zeev-Ben-Mordehai

2015-12-01

Full Text Available Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC, which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

Immersion freezing of ice nucleation active protein complexes

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S. Hartmann

2013-06-01

Full Text Available Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS, the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice

Overcoming barriers to membrane protein structure determination.

Science.gov (United States)

Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

2011-04-01

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Multiscale molecular dynamics simulations of membrane remodeling by Bin/Amphiphysin/Rvs family proteins

Science.gov (United States)

Chun, Chan; Haohua, Wen; Lanyuan, Lu; Jun, Fan

2016-01-01

Membrane curvature is no longer thought of as a passive property of the membrane; rather, it is considered as an active, regulated state that serves various purposes in the cell such as between cells and organelle definition. While transport is usually mediated by tiny membrane bubbles known as vesicles or membrane tubules, such communication requires complex interplay between the lipid bilayers and cytosolic proteins such as members of the Bin/Amphiphysin/Rvs (BAR) superfamily of proteins. With rapid developments in novel experimental techniques, membrane remodeling has become a rapidly emerging new field in recent years. Molecular dynamics (MD) simulations are important tools for obtaining atomistic information regarding the structural and dynamic aspects of biological systems and for understanding the physics-related aspects. The availability of more sophisticated experimental data poses challenges to the theoretical community for developing novel theoretical and computational techniques that can be used to better interpret the experimental results to obtain further functional insights. In this review, we summarize the general mechanisms underlying membrane remodeling controlled or mediated by proteins. While studies combining experiments and molecular dynamics simulations recall existing mechanistic models, concurrently, they extend the role of different BAR domain proteins during membrane remodeling processes. We review these recent findings, focusing on how multiscale molecular dynamics simulations aid in understanding the physical basis of BAR domain proteins, as a representative of membrane-remodeling proteins. Project supported by the National Natural Science Foundation of China (Grant No. 21403182) and the Research Grants Council of Hong Kong, China (Grant No. CityU 21300014).

Malaria Parasite CLAG3, a Protein Linked to Nutrient Channels, Participates in High Molecular Weight Membrane-Associated Complexes in the Infected Erythrocyte.

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Kayvan Zainabadi

Full Text Available Malaria infected erythrocytes show increased permeability to a number of solutes important for parasite growth as mediated by the Plasmodial Surface Anion Channel (PSAC. The P. falciparum clag3 genes have recently been identified as key determinants of PSAC, though exactly how they contribute to channel function and whether additional host/parasite proteins are required remain unknown. To begin to answer these questions, I have taken a biochemical approach. Here I have used an epitope-tagged CLAG3 parasite to perform co-immunoprecipitation experiments using membrane fractions of infected erythrocytes. Native PAGE and mass spectrometry studies reveal that CLAG3 participate in at least three different high molecular weight complexes: a ~720kDa complex consisting of CLAG3, RHOPH2 and RHOPH3; a ~620kDa complex consisting of CLAG3 and RHOPH2; and a ~480kDa complex composed solely of CLAG3. Importantly, these complexes can be found throughout the parasite lifecycle but are absent in untransfected controls. Extracellular biotin labeling and protease susceptibility studies localize the 480kDa complex to the erythrocyte membrane. This complex, likely composed of a homo-oligomer of 160kDa CLAG3, may represent a functional subunit, possibly the pore, of PSAC.

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

Science.gov (United States)

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

Science.gov (United States)

Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

1989-12-01

The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

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Szu-Chia Hsieh

Full Text Available The envelope (E of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

A simple detection method for low-affinity membrane protein interactions by baculoviral display.

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Toshiko Sakihama

Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

Science.gov (United States)

Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

Studying Membrane Protein Structure and Function Using Nanodiscs

DEFF Research Database (Denmark)

Huda, Pie

The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study

DEFF Research Database (Denmark)

Sadaf, Aiman; Du, Yang; Santillan, Claudia

2017-01-01

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein...... alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6...

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

Energy Technology Data Exchange (ETDEWEB)

Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

2017-03-12

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes

International Nuclear Information System (INIS)

Dewa, Takehisa; Sumino, Ayumi; Watanabe, Natsuko; Noji, Tomoyasu; Nango, Mamoru

2013-01-01

Highlights: ► Photosynthetic light-harvesting complexes were reconstituted into lipid membranes. ► Energy transfers between light-harvesting complexes were examined. ► Atomic force microscopy indicated cluster formation of light-harvesting complexes. ► Efficient energy transfer was observed for the clustered complexes in the membranes. - Abstract: In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency

Energy transfer and clustering of photosynthetic light-harvesting complexes in reconstituted lipid membranes

Energy Technology Data Exchange (ETDEWEB)

Dewa, Takehisa, E-mail: takedewa@nitech.ac.jp [Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan); Japan Science and Technology, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012 (Japan); Sumino, Ayumi; Watanabe, Natsuko; Noji, Tomoyasu [Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan); Nango, Mamoru, E-mail: nango@nitech.ac.jp [Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan)

2013-06-20

Highlights: ► Photosynthetic light-harvesting complexes were reconstituted into lipid membranes. ► Energy transfers between light-harvesting complexes were examined. ► Atomic force microscopy indicated cluster formation of light-harvesting complexes. ► Efficient energy transfer was observed for the clustered complexes in the membranes. - Abstract: In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

OpenAIRE

Evan Quon; Christopher T. Beh

2016-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer...

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Science.gov (United States)

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

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.

Protein profiles of hatchery egg shell membrane.

Science.gov (United States)

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

2016-01-01

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

Multi-scaled normal mode analysis method for dynamics simulation of protein-membrane complexes: A case study of potassium channel gating motion correlations

Energy Technology Data Exchange (ETDEWEB)

Wu, Xiaokun; Han, Min; Ming, Dengming, E-mail: dming@fudan.edu.cn [Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai (China)

2015-10-07

Membrane proteins play critically important roles in many cellular activities such as ions and small molecule transportation, signal recognition, and transduction. In order to fulfill their functions, these proteins must be placed in different membrane environments and a variety of protein-lipid interactions may affect the behavior of these proteins. One of the key effects of protein-lipid interactions is their ability to change the dynamics status of membrane proteins, thus adjusting their functions. Here, we present a multi-scaled normal mode analysis (mNMA) method to study the dynamics perturbation to the membrane proteins imposed by lipid bi-layer membrane fluctuations. In mNMA, channel proteins are simulated at all-atom level while the membrane is described with a coarse-grained model. mNMA calculations clearly show that channel gating motion can tightly couple with a variety of membrane deformations, including bending and twisting. We then examined bi-channel systems where two channels were separated with different distances. From mNMA calculations, we observed both positive and negative gating correlations between two neighboring channels, and the correlation has a maximum as the channel center-to-center distance is close to 2.5 times of their diameter. This distance is larger than recently found maximum attraction distance between two proteins embedded in membrane which is 1.5 times of the protein size, indicating that membrane fluctuation might impose collective motions among proteins within a larger area. The hybrid resolution feature in mNMA provides atomic dynamics information for key components in the system without costing much computer resource. We expect it to be a conventional simulation tool for ordinary laboratories to study the dynamics of very complicated biological assemblies. The source code is available upon request to the authors.

Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain.

Directory of Open Access Journals (Sweden)

Jane A English

Full Text Available Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

Lipid Directed Intrinsic Membrane Protein Segregation

DEFF Research Database (Denmark)

Hansen, Jesper S.; Thompson, James R.; Helix Nielsen, Claus

2013-01-01

We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily h...

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

Science.gov (United States)

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

Science.gov (United States)

Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

2017-11-09

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Science.gov (United States)

Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

2013-01-01

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

Disturbed vesicular trafficking of membrane proteins in prion disease.

Science.gov (United States)

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

Science.gov (United States)

Kahle, M; Schäfer, A; Seelig, A; Schultheiß, J; Wu, M; Aichler, M; Leonhardt, J; Rathkolb, B; Rozman, J; Sarioglu, H; Hauck, S M; Ueffing, M; Wolf, E; Kastenmueller, G; Adamski, J; Walch, A; Hrabé de Angelis, M; Neschen, S

2015-01-01

Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and

Self diffusion and spectral modifications of a membrane protein, the Rubrivivax gelatinosus LH2 complex, incorporated into a monoolein cubic phase.

OpenAIRE

Tsapis, N; Reiss-Husson, F; Ober, R; Genest, M; Hodges, R S; Urbach, W

2001-01-01

The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein. We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching. We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its beta-octyl glucoside solution. In these experiments, native LH2 and LH2 labeled ...

Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

Science.gov (United States)

Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

2015-11-01

The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. Copyright © 2015 Elsevier GmbH. All rights reserved.

Organizing membrane-curving proteins: the emerging dynamical picture.

Science.gov (United States)

Simunovic, Mijo; Bassereau, Patricia; Voth, Gregory A

2018-03-30

Lipid membranes play key roles in cells, such as in trafficking, division, infection, remodeling of organelles, among others. The key step in all these processes is creating membrane curvature, typically under the control of many anchored, adhered or included proteins. However, it has become clear that the membrane itself can mediate the interactions among proteins to produce highly ordered assemblies. Computer simulations are ideally suited to investigate protein organization and the dynamics of membrane remodeling at near-micron scales, something that is extremely challenging to tackle experimentally. We review recent computational efforts in modeling protein-caused membrane deformation mechanisms, specifically focusing on coarse-grained simulations. We highlight work that exposed the membrane-mediated ordering of proteins into lines, meshwork, spirals and other assemblies, in what seems to be a very generic mechanism driven by a combination of short and long-ranged forces. Modulating the mechanical properties of membranes is an underexplored signaling mechanism in various processes deserving of more attention in the near future. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dynamic complexity: plant receptor complexes at the plasma membrane.

Science.gov (United States)

Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*

Science.gov (United States)

Zhang, Xi

2015-01-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834

Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.

Science.gov (United States)

Zhang, Xi

2015-09-01

The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

Science.gov (United States)

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Overcoming bottlenecks in the membrane protein structural biology pipeline.

Science.gov (United States)

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

Functional dynamics of cell surface membrane proteins.

Science.gov (United States)

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. Copyright © 2013 Elsevier Inc. All rights reserved.

Integral membrane protein structure determination using pseudocontact shifts

Energy Technology Data Exchange (ETDEWEB)

Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

2015-04-15

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

International Nuclear Information System (INIS)

Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A

2008-01-01

Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property

High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

Energy Technology Data Exchange (ETDEWEB)

Vogel, R F; Linke, K; Teichert, H; Ehrmann, M A [Technische Universitaet Muenchen, Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising (Germany)], E-mail: rudi.vogel@wzw.tum.de

2008-07-15

Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

Science.gov (United States)

Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

2008-07-01

Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

Efficient Overproduction of Membrane Proteins in Lactococcus lactis Requires the Cell Envelope Stress Sensor/Regulator Couple CesSR

Science.gov (United States)

Pinto, Joao P. C.; Kuipers, Oscar P.; Marreddy, Ravi K. R.; Poolman, Bert; Kok, Jan

2011-01-01

Background Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. Methods and Findings We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. Conclusions The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins. PMID:21818275

Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

OpenAIRE

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A.; Saul, Allan; Gerke, Christiane

2014-01-01

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Int...

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Science.gov (United States)

Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

2015-08-18

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

Science.gov (United States)

Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

2011-09-01

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

Detection of proteins on blot transfer membranes.

Science.gov (United States)

Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

Science.gov (United States)

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Imaging and structural studies of DNA–protein complexes and membrane ion channels

KAUST Repository

Marini, Monica; Limongi, Tania; Falqui, Andrea; Genovese, Alessandro; Allione, Marco; Moretti, Manola; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Cesca, F.; Benfenati, F.; Di Fabrizio, Enzo M.

2017-01-01

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABA(A) channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 angstrom, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Imaging and structural studies of DNA–protein complexes and membrane ion channels

KAUST Repository

Marini, Monica

2017-01-17

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABA(A) channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 angstrom, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Challenges in the Development of Functional Assays of Membrane Proteins

Directory of Open Access Journals (Sweden)

Sophie Demarche

2012-11-01

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

Biophysical EPR Studies Applied to Membrane Proteins

Science.gov (United States)

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

Structuring detergents for extracting and stabilizing functional membrane proteins.

Directory of Open Access Journals (Sweden)

Rima Matar-Merheb

Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

Science.gov (United States)

Kilejian, A; Rashid, M A; Aikawa, M; Aji, T; Yang, Y F

1991-02-01

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

Tandem Facial Amphiphiles for Membrane Protein Stabilization

DEFF Research Database (Denmark)

Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer

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....

Low-Resolution Structure of Detergent-Solubilized Membrane Proteins from Small-Angle Scattering Data.

Science.gov (United States)

Koutsioubas, Alexandros

2017-12-05

Despite the ever-increasing usage of small-angle scattering as a valuable complementary method in the field of structural biology, applications concerning membrane proteins remain elusive mainly due to experimental challenges and the relative lack of theoretical tools for the treatment of scattering data. This fact adds up to general difficulties encountered also by other established methods (crystallography, NMR) for the study of membrane proteins. Following the general paradigm of ab initio methods for low-resolution restoration of soluble protein structure from small-angle scattering data, we construct a general multiphase model with a set of physical constraints, which, together with an appropriate minimization procedure, gives direct structural information concerning the different components (protein, detergent molecules) of detergent-solubilized membrane protein complexes. Assessment of the method's precision and robustness is evaluated by performing shape restorations from simulated data of a tetrameric α-helical membrane channel (Aquaporin-0) solubilized by n-Dodecyl β-D-Maltoside and from previously published small-angle neutron scattering experimental data of the filamentous hemagglutinin adhesin β-barrel protein transporter solubilized by n-Octyl β-D-glucopyranoside. It is shown that the acquisition of small-angle neutron scattering data at two different solvent contrasts, together with an estimation of detergent aggregation number around the protein, permits the reliable reconstruction of the shape of membrane proteins without the need for any prior structural information. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Discriminating lysosomal membrane protein types using dynamic neural network.

Science.gov (United States)

Tripathi, Vijay; Gupta, Dwijendra Kumar

2014-01-01

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

Assembly of Photosynthetic Antenna Protein Complexes from Algae for Development of Nano-biodevice and Its Fuelization

Science.gov (United States)

2013-05-20

bacterial photosynthesis . The structure of the reaction center (RC, the first membrane protein to have its structure determined to high resolution...Introduction] In a bacterial photosynthesis , light-harvesting complex 2 (LH2) and lightharvesting-reaction center complex (LH1-RC) play the key...Artificial Leaf 6CO2 + 6H2O C6H12O6 (Glucose) +6O2 Natural Leaf Photosynthesis and redox proteins are well-organized into thylakoid membrane in natural leaf

Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

DEFF Research Database (Denmark)

Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw

2014-01-01

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

Characterization of auxin-binding proteins from zucchini plasma membrane

Science.gov (United States)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

Artificial membranes with selective nanochannels for protein transport

KAUST Repository

Sutisna, Burhannudin

2016-09-05

A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

Artificial membranes with selective nanochannels for protein transport

KAUST Repository

Sutisna, Burhannudin; Polymeropoulos, Georgios; Mygiakis, E.; Musteata, Valentina-Elena; Peinemann, Klaus-Viktor; Smilgies, D. M.; Hadjichristidis, Nikolaos; Nunes, Suzana Pereira

2016-01-01

A poly(styrene-b-tert-butoxystyrene-b-styrene) copolymer was synthesized by anionic polymerization and hydrolyzed to poly(styrene-b-4-hydroxystyrene-b-styrene). Lamellar morphology was confirmed in the bulk after annealing. Membranes were fabricated by self-assembly of the hydrolyzed copolymer in solution, followed by water induced phase separation. A high density of pores of 4 to 5 nm diameter led to a water permeance of 40 L m−2 h−1 bar−1 and molecular weight cut-off around 8 kg mol−1. The morphology was controlled by tuning the polymer concentration, evaporation time, and the addition of imidazole and pyridine to stabilize the terpolymer micelles in the casting solution via hydrogen bond complexes. Transmission electron microscopy of the membrane cross-sections confirmed the formation of channels with hydroxyl groups beneficial for hydrogen-bond forming sites. The morphology evolution was investigated by time-resolved grazing incidence small angle X-ray scattering experiments. The membrane channels reject polyethylene glycol with a molecular size of 10 kg mol−1, but are permeable to proteins, such as lysozyme (14.3 kg mol−1) and cytochrome c (12.4 kg mol−1), due to the right balance of hydrogen bond interactions along the channels, electrostatic attraction, as well as the right pore sizes. Our results demonstrate that artificial channels can be designed for protein transport via block copolymer self-assembly using classical methods of membrane preparation.

Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria.

Science.gov (United States)

Waegemann, Karin; Popov-Čeleketić, Dušan; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

2015-03-13

Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

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

Science.gov (United States)

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

2016-11-01

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

Macrolide Resistance Mediated by a Bifidobacterium breve Membrane Protein

OpenAIRE

Margolles, Abelardo; Moreno, José Antonio; van Sinderen, Douwe; de los Reyes-Gavilán, Clara G.

2005-01-01

A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.

Phytochemicals perturb membranes and promiscuously alter protein function.

Science.gov (United States)

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.

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

International Nuclear Information System (INIS)

Witt, P.L.; Bownds, M.D.

1987-01-01

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

Membrane fusion and exocytosis.

Science.gov (United States)

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

DEFF Research Database (Denmark)

Elortza, Felix; Nühse, Thomas S; Foster, Leonard J

2003-01-01

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

Structural adaptations of proteins to different biological membranes

Science.gov (United States)

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

Alterations in membrane protein-profile during cold treatment of alfalfa

International Nuclear Information System (INIS)

Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S.

1988-01-01

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [ 35 S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo

Dynamic, electronically switchable surfaces for membrane protein microarrays.

Science.gov (United States)

Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

2006-02-01

Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

Heat-induced reorganization of the structure of photosystem II membranes: role of oxygen evolving complex.

Science.gov (United States)

Busheva, Mira; Tzonova, Iren; Stoitchkova, Katerina; Andreeva, Atanaska

2012-12-05

The sensitivity of the green plants' photosystem II (PSII) to high temperatures is investigated in PSII enriched membranes and in membranes, from which the oxygen evolving complex is removed. Using steady-state 77 K fluorescence and resonance Raman spectroscopy we analyze the interdependency between the temperature-driven changes in structure and energy distribution in the PSII supercomplex. The results show that the heat treatment induces different reduction of the 77 K fluorescence emission in both types of investigated membranes: (i) an additional considerable decrease of the overall fluorescence emission in Tris-washed membranes as compared to the native membranes; (ii) a transition point at 42°C(,) observed only in native membranes; (iii) a sharp reduction of the PSII core fluorescence in Tris-washed membranes at temperatures higher than 50°C; (iv) a 3 nm red-shift of F700 band's maximum in Tris-washed membranes already at 20°C and its further shift by 1 nm at temperature increase. Both treatments intensified their action by increasing the aggregation and dissociation of the peripheral light harvesting complexes. The oxygen-evolving complex, in addition to its main function to produce O(2), increases the thermal stability of PSII core by strengthening the connection between the core and the peripheral antenna proteins and by keeping their structural integrity. Copyright © 2012 Elsevier B.V. All rights reserved.

[Adsorption characteristics of proteins on membrane surface and effect of protein solution environment on permeation behavior of berberine].

Science.gov (United States)

Li, Yi-Qun; Xu, Li; Zhu, Hua-Xu; Tang, Zhi-Shu; Li, Bo; Pan, Yong-Lan; Yao, Wei-Wei; Fu, Ting-Ming; Guo, Li-Wei

2017-10-01

In order to explore the adsorption characteristics of proteins on the membrane surface and the effect of protein solution environment on the permeation behavior of berberine, berberine and proteins were used as the research object to prepare simulated solution. Low field NMR, static adsorption experiment and membrane separation experiment were used to study the interaction between the proteins and ceramic membrane or between the proteins and berberine. The static adsorption capacity of proteins, membrane relative flux, rejection rate of proteins, transmittance rate of berberine and the adsorption rate of proteins and berberine were used as the evaluation index. Meanwhile, the membrane resistance distribution, the particle size distribution and the scanning electron microscope (SEM) were determined to investigate the adsorption characteristics of proteins on ceramic membrane and the effect on membrane separation process of berberine. The results showed that the ceramic membrane could adsorb the proteins and the adsorption model was consistent with Langmuir adsorption model. In simulating the membrane separation process, proteins were the main factor to cause membrane fouling. However, when the concentration of proteins was 1 g•L⁻¹, the proteins had no significant effect on membrane separation process of berberine. Copyright© by the Chinese Pharmaceutical Association.

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

OpenAIRE

Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette; Chae, Pil Seok

2016-01-01

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation.

Science.gov (United States)

Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

2016-10-04

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

Science.gov (United States)

Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette

2017-01-01

A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research. PMID:27711401

Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

Science.gov (United States)

Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

2017-09-01

Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

Science.gov (United States)

Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

2014-01-01

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

2009-01-01

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane...... purification and comparative quantitative LC-MS/MS proteomic analysis identified 13 membrane proteins that were expressed at higher levels and 3 that were under-expressed in the metastatic compared to the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5...

Membrane Proteins : The Key Players of a Cancer Cell

NARCIS (Netherlands)

Kampen, Kim R.

Membrane proteins are involved in the prognosis of the most common forms of cancer. Membrane proteins are the hallmark of a cancer cell. The overexpressed membrane receptors are becoming increasingly important in cancer cell therapy. Current renewing therapy approaches based on receptor

Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

International Nuclear Information System (INIS)

Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

1987-01-01

The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes.

NARCIS (Netherlands)

Wessels, H.C.T.; Vogel, R.O.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.; Rodenburg, R.J.T.; Nijtmans, L.G.J.; Farhoud, M.H.

2009-01-01

Two-dimensional blue native/SDS-PAGE is widely applied to investigate native protein-protein interactions, particularly those within membrane multi-protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we

A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria

NARCIS (Netherlands)

Boekema, E.J.; Hifney, A.; Yakushevska, A.E.; Piotrowski, M.; Keegstra, W.; Berry, S.; Michel, K.-P.; Pistorius, E.K.; Kruip, J.

2001-01-01

Cyanobacteria are abundant throughout most of the world's water bodies and contribute significantly to global primary productivity through oxygenic photosynthesis. This reaction is catalysed by two membrane-bound protein complexes, photosystem I (PSI) and photosystem II (PSII), which both contain

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

Science.gov (United States)

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Cryo-electron microscopy of membrane proteins.

Science.gov (United States)

Goldie, Kenneth N; Abeyrathne, Priyanka; Kebbel, Fabian; Chami, Mohamed; Ringler, Philippe; Stahlberg, Henning

2014-01-01

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Role for chlamydial inclusion membrane proteins in inclusion membrane structure and biogenesis.

Directory of Open Access Journals (Sweden)

Jeffrey Mital

Full Text Available 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.

Major Intrinsic Proteins in Biomimetic Membranes

DEFF Research Database (Denmark)

Helix Nielsen, Claus

2010-01-01

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....../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 109 molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other...

A New Strain Collection for Improved Expression of Outer Membrane Proteins

Directory of Open Access Journals (Sweden)

Ina Meuskens

2017-11-01

Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

PredMP: A Web Resource for Computationally Predicted Membrane Proteins via Deep Learning

KAUST Repository

Wang, Sheng

2018-02-06

Experimental determination of membrane protein (MP) structures is challenging as they are often too large for nuclear magnetic resonance (NMR) experiments and difficult to crystallize. Currently there are only about 510 non-redundant MPs with solved structures in Protein Data Bank (PDB). To elucidate the MP structures computationally, we developed a novel web resource, denoted as PredMP (http://52.87.130.56:3001/#/proteinindex), that delivers one-dimensional (1D) annotation of the membrane topology and secondary structure, two-dimensional (2D) prediction of the contact/distance map, together with three-dimensional (3D) modeling of the MP structure in the lipid bilayer, for each MP target from a given model organism. The precision of the computationally constructed MP structures is leveraged by state-of-the-art deep learning methods as well as cutting-edge modeling strategies. In particular, (i) we annotate 1D property via DeepCNF (Deep Convolutional Neural Fields) that not only models complex sequence-structure relationship but also interdependency between adjacent property labels; (ii) we predict 2D contact/distance map through Deep Transfer Learning which learns the patterns as well as the complex relationship between contacts/distances and protein features from non-membrane proteins; and (iii) we model 3D structure by feeding its predicted contacts and secondary structure to the Crystallography & NMR System (CNS) suite combined with a membrane burial potential that is residue-specific and depth-dependent. PredMP currently contains more than 2,200 multi-pass transmembrane proteins (length<700 residues) from Human. These transmembrane proteins are classified according to IUPHAR/BPS Guide, which provides a hierarchical organization of receptors, channels, transporters, enzymes and other drug targets according to their molecular relationships and physiological functions. Among these MPs, we estimated that our approach could predict correct folds for 1

Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.

Science.gov (United States)

Yang, Yoosoo; Hong, Yeonsun; Cho, Eunji; Kim, Gi Beom; Kim, In-San

2018-01-01

Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.

Biosurfactants and surfactants interacting with membranes and proteins: Same but different?

Science.gov (United States)

Otzen, Daniel E

2017-04-01

Biosurfactants (BS) are surface-active molecules produced by microorganisms. For several decades they have attracted interest as promising alternatives to current petroleum-based surfactants. Aside from their green profile, they have remarkably low critical micelle concentrations, reduce the air/water surface tension to very low levels and are excellent emulsifiers, all of which make them comparable or superior to their synthetic counterparts. These remarkable physical properties derive from their more complex chemical structures in which hydrophilic and hydrophobic regions are not as clearly separated as chemical surfactants but have a more mosaic distribution of polarity as well as branched or circular structures. This allows the lipopeptide surfactin to adopt spherical structures to facilitate dense packing at interfaces. They are also more complex. Glycolipid BS, e.g. rhamnolipids (RL) and sophorolipids, are produced biologically as mixtures which vary in the size and saturation of the hydrophobic region as well as modifications in the hydrophilic headgroup, such as the number of sugar groups and different levels of acetylation, leading to variable surface-active properties. Their amphiphilicity allows RL to insert easily into membranes at sub-cmc concentrations to modulate membrane structure and extract lipopolysaccharides, leading to extensive biofilm remodeling in vivo, sometimes in collaboration with hydrophobic RL precursors. Thanks to their mosaicity, even anionic BS like RL only bind weakly to proteins and show much lower denaturing potency, even supporting membrane protein refolding. Nevertheless, they can promote protein degradation by proteases e.g. by neutralizing positive charges, which together with their biofilm-combating properties makes them very promising detergent surfactants. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016 Elsevier B

Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition.

Science.gov (United States)

Hayat, Maqsood; Khan, Asifullah

2011-02-21

Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor. Copyright © 2010 Elsevier Ltd. All rights reserved.

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

Science.gov (United States)

Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

2010-02-01

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

Science.gov (United States)

Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

2015-08-04

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes

International Nuclear Information System (INIS)

Caffrey, Martin

2015-01-01

A comprehensive and up-to-date review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes is reported. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes

Energy Technology Data Exchange (ETDEWEB)

Caffrey, Martin, E-mail: martin.caffrey@tcd.ie [Trinity College Dublin, Dublin (Ireland)

2015-01-01

A comprehensive and up-to-date review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes is reported. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for

Crystal structure of the potassium-importing KdpFABC membrane complex

Energy Technology Data Exchange (ETDEWEB)

Huang, Ching-Shin; Pedersen, Bjørn Panyella; Stokes, David L.

2017-06-21

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting1. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.

Membrane Protein Production in Lactococcus lactis for Functional Studies.

Science.gov (United States)

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

2016-01-01

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

Regulation of multispanning membrane protein topology via post-translational annealing.

Science.gov (United States)

Van Lehn, Reid C; Zhang, Bin; Miller, Thomas F

2015-09-26

The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

NMR structural studies of peptides and proteins in membranes

Energy Technology Data Exchange (ETDEWEB)

Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

1994-12-31

The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.

The fifth adaptor protein complex.

Directory of Open Access Journals (Sweden)

Jennifer Hirst

2011-10-01

Full Text Available Adaptor protein (AP complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

Monitoring Protein Fouling on Polymeric Membranes Using Ultrasonic Frequency-Domain Reflectometry

Directory of Open Access Journals (Sweden)

Robin Fong

2011-08-01

Full Text Available Novel signal-processing protocols were used to extend the in situ sensitivity of ultrasonic frequency-domain reflectometry (UFDR for real-time monitoring of microfiltration (MF membrane fouling during protein purification. Different commercial membrane materials, with a nominal pore size of 0.2 µm, were challenged using bovine serum albumin (BSA and amylase as model proteins. Fouling induced by these proteins was observed in flat-sheet membrane filtration cells operating in a laminar cross-flow regime. The detection of membrane-associated proteins using UFDR was determined by applying rigorous statistical methodology to reflection spectra of ultrasonic signals obtained during membrane fouling. Data suggest that the total power reflected from membrane surfaces changes in response to protein fouling at concentrations as low as 14 μg/cm2, and results indicate that ultrasonic spectra can be leveraged to detect and monitor protein fouling on commercial MF membranes.

Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

DEFF Research Database (Denmark)

Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde

2012-01-01

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps....../periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli....

Self-assembling peptides form nanodiscs that stabilize membrane proteins

DEFF Research Database (Denmark)

Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

2014-01-01

-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

Directory of Open Access Journals (Sweden)

Sylvie Lalonde

2010-09-01

Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

Science.gov (United States)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

Science.gov (United States)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

Science.gov (United States)

Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

2017-08-22

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

Science.gov (United States)

Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

2018-01-05

, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

Analysis of protein-protein docking decoys using interaction fingerprints: application to the reconstruction of CaM-ligand complexes

Directory of Open Access Journals (Sweden)

Uchikoga Nobuyuki

2010-05-01

Full Text Available Abstract Background Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles. Results To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG, CaM kinase kinase (CaMKK and the plasma membrane Ca2+ ATPase pump (PMCA, and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK. Conclusions The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases.

Synthesis and Functional Reconstitution of Light-Harvesting Complex II into Polymeric Membrane Architectures.

Science.gov (United States)

Zapf, Thomas; Tan, Cherng-Wen Darren; Reinelt, Tobias; Huber, Christoph; Shaohua, Ding; Geifman-Shochat, Susana; Paulsen, Harald; Sinner, Eva-Kathrin

2015-12-01

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light-harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar-energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer-based membrane systems containing LHCII-pigment complexes ready for light harvesting. LHCII was produced by cell-free protein synthesis based on wheat-germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

Directory of Open Access Journals (Sweden)

Bin Lu

2015-06-01

Full Text Available Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2 on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26 abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9 loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

Science.gov (United States)

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

Science.gov (United States)

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

International Nuclear Information System (INIS)

Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

2006-01-01

The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Science.gov (United States)

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

The dynamics of plant plasma membrane proteins: PINs and beyond.

Science.gov (United States)

Luschnig, Christian; Vert, Grégory

2014-08-01

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement

DEFF Research Database (Denmark)

Kublik, Anja; Deobald, Darja; Hartwig, Stefanie

2016-01-01

electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250–270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster...... of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi....

Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

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Pitter F Huesgen

Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

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Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

2017-01-01

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Functional discrimination of membrane proteins using machine learning techniques

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Yabuki Yukimitsu

2008-03-01

Full Text Available Abstract Background Discriminating membrane proteins based on their functions is an important task in genome annotation. In this work, we have analyzed the characteristic features of amino acid residues in membrane proteins that perform major functions, such as channels/pores, electrochemical potential-driven transporters and primary active transporters. Results We observed that the residues Asp, Asn and Tyr are dominant in channels/pores whereas the composition of hydrophobic residues, Phe, Gly, Ile, Leu and Val is high in electrochemical potential-driven transporters. The composition of all the amino acids in primary active transporters lies in between other two classes of proteins. We have utilized different machine learning algorithms, such as, Bayes rule, Logistic function, Neural network, Support vector machine, Decision tree etc. for discriminating these classes of proteins. We observed that most of the algorithms have discriminated them with similar accuracy. The neural network method discriminated the channels/pores, electrochemical potential-driven transporters and active transporters with the 5-fold cross validation accuracy of 64% in a data set of 1718 membrane proteins. The application of amino acid occurrence improved the overall accuracy to 68%. In addition, we have discriminated transporters from other α-helical and β-barrel membrane proteins with the accuracy of 85% using k-nearest neighbor method. The classification of transporters and all other proteins (globular and membrane showed the accuracy of 82%. Conclusion The performance of discrimination with amino acid occurrence is better than that with amino acid composition. We suggest that this method could be effectively used to discriminate transporters from all other globular and membrane proteins, and classify them into channels/pores, electrochemical and active transporters.

Dissection of membrane protein degradation mechanisms by reversible inhibitors

International Nuclear Information System (INIS)

Hare, J.F.

1988-01-01

The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma membrane polypeptides in response to reversible temperature and lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were radiolabeled and left for 24 h to allow the removal of rapidly degraded proteins. Remaining trichloroacetic acid-precipitable protein was degraded (t 1/2 = 40-68 h) by an apparent first order process 60-86% sensitive to 10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by temperature reduction to 18 degrees C. Thus, membrane proteins are selected for degradation in a time-dependent manner by a system which is sensitive to both 18 degrees C and to lysosomotropic amines. When inhibitory conditions were removed after 40-48 h, degradation of 125I-labeled protein resumed at the same rate as that seen in their absence. Since membrane proteins do not exhibit accelerated degradation after removal of inhibitory conditions, there can be no marking or sorting of those proteins destined for degradation during the 40-h exposure to inhibitory conditions. Exposure to amines or 18 degrees C did not affect the position of two-dimensionally resolved labeled polypeptides. Fractionation of labeled cells on Percoll gradients after 40 h of exposure to low temperature or amines showed that labeled protein remained in the plasma membrane fractions of the gradient although shifted to a slightly lower buoyant density in the presence of amines. These results support the notion that selection of plasma membrane proteins for degradation requires their internalization into acidic vesicles. Lysosomotropic amines and reduced temperature interfere with the selection process by preventing membrane fusion events

Analysis of protein interactions at native chloroplast membranes by ellipsometry.

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Verena Kriechbaumer

Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

Science.gov (United States)

Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

2014-01-01

Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

Protein secretion and membrane insertion systems in gram-negative bacteria.