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Sample records for membrane protein studies

  1. Thermostabilisation of membrane proteins for structural studies

    PubMed Central

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

    2017-01-01

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

  2. Engineering Lipid Bilayer Membranes for Protein Studies

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Straatsma, TP

    2006-12-01

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

  4. Novel benzanthrone probes for membrane and protein studies

    NASA Astrophysics Data System (ADS)

    Ryzhova, Olga; Vus, Kateryna; Trusova, Valeriya; Kirilova, Elena; Kirilov, Georgiy; Gorbenko, Galyna; Kinnunen, Paavo

    2016-09-01

    The applicability of a series of novel benzanthrone dyes to monitoring the changes in physicochemical properties of lipid bilayer and to differentiating between the native and aggregated protein states has been evaluated. Based on the quantitative parameters of the dye-membrane and dye-protein binding derived from the fluorimetric titration data, the most prospective membrane probes and amyloid tracers have been selected from the group of examined compounds. Analysis of the red edge excitation shifts of the membrane- and amyloid-bound dyes provided information on the properties of benzanthrone binding sites within the lipid and protein matrixes. To understand how amyloid specificity of benzanthrones correlates with their structure, quantitative structure activity relationship (QSAR) analysis was performed involving a range of quantum chemical molecular descriptors. A statistically significant model was obtained for predicting the sensitivity of novel benzanthrone dyes to amyloid fibrils.

  5. The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations.

    PubMed

    Grouleff, Julie; Irudayam, Sheeba Jem; Skeby, Katrine K; Schiøtt, Birgit

    2015-09-01

    The plasma membrane, which encapsulates human cells, is composed of a complex mixture of lipids and embedded proteins. Emerging knowledge points towards the lipids as having a regulating role in protein function. Furthermore, insight from protein crystallography has revealed several different types of lipids intimately bound to membrane proteins and peptides, hereby possibly pointing to a site of action for the observed regulation. Cholesterol is among the lipid membrane constituents most often observed to be co-crystallized with membrane proteins, and the cholesterol levels in cell membranes have been found to play an essential role in health and disease. Remarkably little is known about the mechanism of lipid regulation of membrane protein function in health as well as in disease. Herein, we review molecular dynamics simulation studies aimed at investigating the effect of cholesterol on membrane protein and peptide properties. This article is part of a Special Issue entitled: Lipid-protein interactions.

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

    PubMed

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

    2014-10-21

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

  7. Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine.

    PubMed

    Kamijo, Akio; Saitoh, Yurika; Ohno, Nobuhiko; Ohno, Shinichi; Terada, Nobuo

    2016-01-01

    The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

  8. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    NASA Astrophysics Data System (ADS)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  9. The synthesis of recombinant membrane proteins in yeast for structural studies.

    PubMed

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

  10. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies

    PubMed Central

    1984-01-01

    As the spike proteins of Semliki Forest virus (SFV) pass from their site of synthesis in the endoplasmic reticulum (ER) to the cell surface, they must be concentrated and freed from endogenous proteins. To determine the magnitude of this sorting process we have measured the density of spike proteins in membranes of the intracellular transport pathway. In this first paper, using stereological procedures, we have estimated the surface areas of the ER, Golgi complex, and plasma membrane of infected and mock-infected baby hamster kidney cells. First, we estimated the mean cell volume in absolute units. This was done using a novel in situ method which is described in detail. Infection by SFV was found to have no effect on any of the parameters measured. In the accompanying paper ( Quinn , P., G. Griffiths, and G. Warren, 1984, J. Cell Biol., 2142-2147) these stereological estimates were combined with biochemical estimates of the amount of spike proteins in ER, Golgi complex, and plasma membrane to determine the density in the membranes of these compartments. PMID:6563037

  11. Atomic force microscopy: a multifaceted tool to study membrane proteins and their interactions with ligands.

    PubMed

    Whited, Allison M; Park, Paul S-H

    2014-01-01

    Membrane proteins are embedded in lipid bilayers and facilitate the communication between the external environment and the interior of the cell. This communication is often mediated by the binding of ligands to the membrane protein. Understanding the nature of the interaction between a ligand and a membrane protein is required to both understand the mechanism of action of these proteins and for the development of novel pharmacological drugs. The highly hydrophobic nature of membrane proteins and the requirement of a lipid bilayer for native function have hampered the structural and molecular characterizations of these proteins under physiologically relevant conditions. Atomic force microscopy offers a solution to studying membrane proteins and their interactions with ligands under physiologically relevant conditions and can provide novel insights about the nature of these critical molecular interactions that facilitate cellular communication. In this review, we provide an overview of the atomic force microscopy technique and discuss its application in the study of a variety of questions related to the interaction between a membrane protein and a ligand. This article is part of a Special Issue entitled: Structural and biophysical characterization of membrane protein-ligand binding.

  12. Tracking membrane protein association in model membranes.

    PubMed

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

    2009-01-01

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

  13. Proteins causing membrane fouling in membrane bioreactors.

    PubMed

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

    2015-01-01

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

  14. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    SciTech Connect

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  15. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  16. A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

    PubMed Central

    Sadaf, Aiman; Mortensen, Jonas S.; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette

    2015-01-01

    Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science. PMID:27110345

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

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

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

  18. Electron paramagnetic resonance studies of proteins in ozone-treated erythrocyte membranes.

    PubMed

    Wróbel, A; Jezierski, A; Gomułkiewicz, J

    1997-02-01

    The sulfhydryl group specific spin labels have been used to study the membrane proteins in the bovine and human erythrocyte membranes treated with ozone. The ratio hw/hs, determined from the respective peak amplitudes of the resulting EPR spectra of 3-maleimido-1-oxyl-2,2,5,5-tetramethylpyrrolidine bound to the ozone treated membranes, was shown to be elevated following an increased ozone concentration. The ratio of the central to high-field peak amplitude h0/h-1, a measure of the rotational correlation time, determined from EPR spectra of ozonized membranes covalently labeled with 4-(iodoacetamido)-1-oxyl-2,2,5, 5-tetramethylpyrrolidine was shown to be diminished. The changes in the erythrocyte membranes treated with ozone, observed for both spin labels, indicate a dose dependent increase in the mobility of membrane proteins.

  19. NMR-based simulation studies of Pf1 coat protein in explicit membranes.

    PubMed

    Cheng, Xi; Jo, Sunhwan; Marassi, Francesca M; Im, Wonpil

    2013-08-06

    As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.

  20. How some proteins tubulate membranes

    NASA Astrophysics Data System (ADS)

    Bassereau, Patricia

    2009-03-01

    Endocytosis, exocytosis, membrane transport between intracellular compartments, virus or toxin entry or exit out of the cell, all imply to deform membrane. Membrane deformation mechanisms of cell membranes by proteins are currently actively studied. Giant vesicles (GUV) are interesting model membrane systems because they are composed of a very limited number of components compared to cellular membranes. The deformations induced by the interaction with a specific protein or any other additional components to the system, can then be directly monitored and the deformation mechanism eventually understood. In this talk, we will focus on different tubular structures induced by proteins. We will show that the B-subunits of Shiga toxin or Cholera Toxin, binding to their lipid receptors, Gb3 or GM1 respectively, incorporated in GUV membrane, induce negative membrane curvature and form tubular invaginations, in absence of any other cellular machinery. Tubular structures can also be obtained when molecular motors walking along microtubules exert a pulling force on the membrane of GUV. The helicoidal assembly of dynamin, a protein involved in vivo in membrane fission can also produce tubular structures. This assembly has been reconstituted around membrane nanotubes of controlled diameter; we will show that the initial tube diameter strongly influences dynamin polymerisation. In each case, a physical framework for understanding deformation mechanism will be presented

  1. Single-particle electron microscopy in the study of membrane protein structure.

    PubMed

    De Zorzi, Rita; Mi, Wei; Liao, Maofu; Walz, Thomas

    2016-02-01

    Single-particle electron microscopy (EM) provides the great advantage that protein structure can be studied without the need to grow crystals. However, due to technical limitations, this approach played only a minor role in the study of membrane protein structure. This situation has recently changed dramatically with the introduction of direct electron detection device cameras, which allow images of unprecedented quality to be recorded, also making software algorithms, such as three-dimensional classification and structure refinement, much more powerful. The enhanced potential of single-particle EM was impressively demonstrated by delivering the first long-sought atomic model of a member of the biomedically important transient receptor potential channel family. Structures of several more membrane proteins followed in short order. This review recounts the history of single-particle EM in the study of membrane proteins, describes the technical advances that now allow this approach to generate atomic models of membrane proteins and provides a brief overview of some of the membrane protein structures that have been studied by single-particle EM to date.

  2. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

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

  3. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

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

  4. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGES

    Johs, Alexander; Whited, A. M.

    2015-01-01

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

  5. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

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

  6. Structural Studies on Membrane Proteins and Biological Macromolecular Assemblies in Japan

    NASA Astrophysics Data System (ADS)

    Tsukihara, Tomitake

    Structural studies on membrane proteins have been performed at atomic level by both three-dimensional X-ray crystallography and two-dimensional electron crystallography in Japan as in Europe and Unites States. More than 13 membrane protein structures were elucidate by X-ray method in our country, and seven membrane protein structures were determined by cryo-electron microscopic method developed by Fujiyoshi of Kyoto University. Extensive crystallographic studies on calcium pump and cytochrome c oxidase elucidated their functional mechanisms at atomic level. Structure and switching mechanism of a flagellum were studied by X-ray and electron microscopic methods. Vault structure exhibiting D39 symmetry was determined by X-ray method.

  7. Lipid-protein interactions of integral membrane proteins: a comparative simulation study.

    PubMed

    Deol, Sundeep S; Bond, Peter J; Domene, Carmen; Sansom, Mark S P

    2004-12-01

    The interactions between membrane proteins and their lipid bilayer environment play important roles in the stability and function of such proteins. Extended (15-20 ns) molecular dynamics simulations have been used to explore the interactions of two membrane proteins with phosphatidylcholine bilayers. One protein (KcsA) is an alpha-helix bundle and embedded in a palmitoyl oleoyl phosphatidylcholine bilayer; the other (OmpA) is a beta-barrel outer-membrane protein and is in a dimyristoyl phosphatidylcholine bilayer. The simulations enable analysis in detail of a number of aspects of lipid-protein interactions. In particular, the interactions of aromatic amphipathic side chains (i.e., Trp, Tyr) with lipid headgroups, and "snorkeling" interactions of basic side chains (i.e., Lys, Arg) with phosphate groups are explored. Analysis of the number of contacts and of H-bonds reveal fluctuations on an approximately 1- to 5-ns timescale. There are two clear bands of interacting residues on the surface of KcsA, whereas there are three such bands on OmpA. A large number of Arg-phosphate interactions are seen for KcsA; for OmpA, the number of basic-phosphate interactions is smaller and shows more marked fluctuations with respect to time. Both classes of interaction occur in clearly defined interfacial regions of width approximately 1 nm. Analysis of lateral diffusion of lipid molecules reveals that "boundary" lipid molecules diffuse at about half the rate of bulk lipid. Overall, these simulations present a dynamic picture of lipid-protein interactions: there are a number of more specific interactions but even these fluctuate on an approximately 1- to 5-ns timescale.

  8. Highly branched penta-saccharide-bearing amphiphiles for membrane protein studies

    PubMed Central

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

    2016-01-01

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

  9. Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins

    PubMed Central

    Kucharska, Iga; Edrington, Thomas C.; Liang, Binyong

    2015-01-01

    Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs. PMID:25869397

  10. Structural Symmetry in Membrane Proteins.

    PubMed

    Forrest, Lucy R

    2015-01-01

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

  11. Proteins of Excitable Membranes

    PubMed Central

    Nachmansohn, David

    1969-01-01

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

  12. Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins.

    PubMed

    Zoonens, Manuela; Giusti, Fabrice; Zito, Francesca; Popot, Jean-Luc

    2007-09-11

    Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep membrane proteins (MPs) water-soluble while stabilizing them biochemically. We have examined the factors that determine the size and dispersity of MP/APol complexes and studied the dynamics of the association, taking as a model system the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) trapped by A8-35, a polyacrylate-based APol. Molecular sieving indicates that the solution properties of the APol largely determine those of tOmpA/APol complexes. Achieving monodispersity depends on using amphipols that themselves form monodisperse particles, on working in neutral or basic solutions, and on the presence of free APols. In order to investigate the role of the latter, a fluorescently labeled version of A8-35 has been synthesized. Förster resonance energy transfer measurements show that extensive dilution of tOmpA/A8-35 particles into an APol-free medium does not entail any detectable desorption of A8-35, even after extended periods of time (hours-days). The fluorescent APol, on the other hand, readily exchanges for other surfactants, be they detergent or unlabeled APol. These findings are discussed in the contexts of sample optimization for MP solution studies and of APol-mediated MP functionalization.

  13. Integrated atomic force microscopy techniques for analysis of biomaterials: Study of membrane proteins

    NASA Astrophysics Data System (ADS)

    Connelly, Laura S.

    Atomic Force Microscopy (AFM) is the prominent techniques for structural studies of biological materials in physiological relevant fluidic environments. AFM has been used to resolve the three-dimensional (3D) surface structure of cells, membranes, and proteins structures. Ion channels, formed by membrane proteins, are the key structures that control the activity of all living systems. This dissertation focuses on the structural evaluation of membrane proteins through atomic force microscopy. In Part I, AFM is utilized to study one of the most prominent medical issues facing our society, Alzheimer's Disease (AD). AD is a misfolded protein disease characterized by the accumulation of beta-amyloid (Abeta) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic channel structures composed of Abeta peptides. Selectively engineered sequences of Abeta were examined by AFM to elucidate the substructures and thus activity Abeta channels. Key residues were evaluated with the intent better understand the exact nature by which these pores conduct electrical and molecular signals, which could aid in identifying potential therapeutic targets for the prevention/treatment of AD. Additionally, AFM was used to analyze brain derived Abeta and newly developed pharmacological agents to study membranes and Abeta. Part II, presents a novel technology that incorporates electrophysiology into the AFM interface, enabling simultaneous imaging and complementary conductance measurements. The activity of ion channels is studied by various techniques, including patch clamp, free standing lipid bilayers, droplet interface bilayers, and supported lipid bilayers. However, direct correlation with channel structures has remained a challenge. The integrated atomic force microscopy system presented offers a solution to this challenge. The functionality of the

  14. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

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

    PubMed Central

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

    2014-01-01

    description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies. PMID:25484487

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

  17. Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

    SciTech Connect

    Haskins, William E.; Leavell, Michael D.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J.; Wood, Nichole L.; Schoeniger, Joseph S.; Kruppa, Gary Hermann; Sale, Kenneth L.; Young, Malin M.; Novak, Petr

    2005-03-01

    Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.

  18. Membrane stiffness is modified by integral membrane proteins.

    PubMed

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

    2016-09-20

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

  19. Studies on the expression of outer membrane protein 2 in escherichia coli.

    PubMed

    Fralick, J A; Diedrich, D L

    1982-01-01

    The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

  20. Membrane proteins: always an insoluble problem?

    PubMed Central

    Rawlings, Andrea E.

    2016-01-01

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

  1. Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system

    PubMed Central

    Niwa, Tatsuya; Sasaki, Yoshihiro; Uemura, Eri; Nakamura, Shugo; Akiyama, Minato; Ando, Mitsuru; Sawada, Shinichi; Mukai, Sada-atu; Ueda, Takuya; Taguchi, Hideki; Akiyoshi, Kazunari

    2015-01-01

    Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins. PMID:26667602

  2. Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system.

    PubMed

    Niwa, Tatsuya; Sasaki, Yoshihiro; Uemura, Eri; Nakamura, Shugo; Akiyama, Minato; Ando, Mitsuru; Sawada, Shinichi; Mukai, Sada-atu; Ueda, Takuya; Taguchi, Hideki; Akiyoshi, Kazunari

    2015-12-15

    Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.

  3. Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.

    PubMed

    Rues, Ralf-Bernhardt; Henrich, Erik; Boland, Coilin; Caffrey, Martin; Bernhard, Frank

    2016-01-01

    The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.

  4. Study of the enhancement effect of cyclopentadecanolide on protein permeation through lipid membranes

    NASA Astrophysics Data System (ADS)

    Li, Zhengmao

    Intranasal drug delivery has been a topic of increasing interest for a decade as a convenient and reliable method for the systemic administration of drugs. The low bioavailability of simple formulation of protein drugs, such as insulin, can be greatly improved by using permeation enhancers. We studied the effect of cyclopentadecanolide (CPE-215RTM) as a permeation enhancer in protein release through lipid bilayer membranes. We successfully designed a novel in-vitro membrane permeability model using liposomes and performed a series of transmembrane protein release experiments. These were carried out under a wide range of conditions in the presence of different permeation enhancer combinations. The experimental results showed that CPE-215RTM is an effective membrane permeation enhancer for proteins and a phase transfer agent, for example, cyclodextrins, can further enhance the effect of CPE-215RTM. Besides the release experiments, studies on insulin solution properties (self-diffusion and self-association states), the interaction between insulin and liposome and the interaction between CPE-215RTM and liposomes were carried out. Based on the mechanistic study and release data, we hypothesized that CPE-215RTM can form transient "pores" in the lipid bilayer that dissolve when CPE-215RTM distributes homogeneously within the bilayer and restore the barrier function of the lipid bilayer. We performed several experiments that corroborate our hypothesis. A mathematical model was developed based on our hypothesized release mechanism. A semi-empirical nonlinear equation involving four parameters effectively fits the protein release profiles. The quality of the data fit with this model is good supporting evidence for the validity of our mechanistic model. Finally we used a neural network approach to correlate the different release condition parameters and the four semi-empirical fitting parameters based on our limited data sets. Reasonable neural networks were formed for the

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

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

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

  6. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

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

  7. The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

    PubMed Central

    Postis, Vincent; Rawson, Shaun; Mitchell, Jennifer K.; Lee, Sarah C.; Parslow, Rosemary A.; Dafforn, Tim R.; Baldwin, Stephen A.; Muench, Stephen P.

    2015-01-01

    Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving > 3.5 Å resolution detail in membrane proteins of modest (~ 300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function. PMID:25450810

  8. Cryoprotection of Lipid Membranes for High-Resolution Solid-State NMR Studies of Membrane Peptides and Proteins at Low Temperature

    PubMed Central

    Lee, Myungwoon; Hong, Mei

    2014-01-01

    Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H MAS spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work – DMSO, PEG, and DMF - should be useful for low

  9. Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature.

    PubMed

    Lee, Myungwoon; Hong, Mei

    2014-08-01

    Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work-DMSO, PEG, and DMF-should be useful for low

  10. Methods for Studying Interactions of Detergents and Lipids withα-Helical and β-Barrel Integral Membrane Proteins

    PubMed Central

    Hasan, S. Saif; Baniulis, Danas; Yamashita, Eiki; Zhalnina, Mariya V.; Zakharov, Stanislav D.; Stofleth, Jason T.; Cramer, William A.

    2014-01-01

    Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical heterooligomeric integral cytochrome b6f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane β-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6f complex, are described. Differences in detergent strategies for isolation and crystallization of β-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented. PMID:24510648

  11. Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties.

    PubMed

    Inglis, Steven R; Turner, Jennifer J; Harding, Margaret M

    2006-12-01

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.

  12. Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants.

    PubMed

    Vertommen, Annelies; Panis, Bart; Swennen, Rony; Carpentier, Sebastien Christian

    2010-04-01

    Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.

  13. Design of membrane proteins: toward functional systems.

    PubMed

    Ghirlanda, Giovanna

    2009-12-01

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

  14. Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p

    SciTech Connect

    Josyula, Ratnakar; Jin, Zhongmin; McCombs, Deborah; DeLucas, Lawrence; Sha, Bingdong

    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.

  15. Zooming in on disordered systems: Neutron reflection studies of proteins associated with fluid membranes

    PubMed Central

    Heinrich, Frank; Lösche, Mathias

    2014-01-01

    Neutron reflectometry (NR) is an emerging experimental technique for the structural characterization of proteins interacting with fluid bilayer membranes under conditions that mimic closely the cellular environment. Thus, cellular processes can be emulated in artificial systems and their molecular basis studied by adding cellular components one at a time in a well-controlled environment while the resulting structures, or structural changes in response to external cues, are monitored with neutron reflection. In recent years, sample environments, data collection strategies and data analysis were continuously refined. The combination of these improvements increases the information which can be obtained from NR to an extent that enables structural characterization of protein-membrane complexes at a length scale that exceeds the resolution of the measurement by far. Ultimately, the combination of NR with molecular dynamics (MD) simulations can be used to cross-validate the results of the two techniques and provide atomicscale structural models. This review discusses these developments in detail and demonstrates how they provide new windows into relevant biomedical problems. PMID:24674984

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

    PubMed Central

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

    1977-01-01

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

  17. Expression and purification of the recombinant membrane protein YidC: a case study for increased stability and solubility.

    PubMed

    Martinez Molina, Daniel; Lundbäck, Anna-Karin; Niegowski, Damian; Eshaghi, Said

    2008-11-01

    YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 microg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies.

  18. NMR study of a membrane protein in detergent-free aqueous solution.

    PubMed

    Zoonens, Manuela; Catoire, Laurent J; Giusti, Fabrice; Popot, Jean-Luc

    2005-06-21

    One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.

  19. Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

    PubMed Central

    Le Brun, Anton P.; Haigh, Cathryn L.; Drew, Simon C.; James, Michael; Boland, Martin P.; Collins, Steven J.

    2014-01-01

    The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions. PMID:25418300

  20. Synthesis and properties of dodecyl trehaloside detergents for membrane protein studies.

    PubMed

    Tao, Houchao; Fu, Yu; Thompson, Aaron; Lee, Sung Chang; Mahoney, Nicholas; Stevens, Raymond C; Zhang, Qinghai

    2012-07-31

    Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization, and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carbon alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC), and size of micelles, were evaluated and compared with the most popular maltoside analogue, β-D-dodecyl maltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive, given their interesting and unique chemical-physical properties and potential interactions with membrane lipids.

  1. A Mössbauer spectroscopy study of the conformational dynamics of native membrane proteins

    NASA Astrophysics Data System (ADS)

    Aleksandrov, A. Yu.; Novakova, A. A.; Semin, B. K.

    1987-07-01

    In a Mössbauer study of non-dehydrated membrane proteins from thermophilic green-blue algae we observed the behavior of the “narrow” and “broad” lines of their Mössbauer spectra during the heating from 80 to 270 K and found that the behavior of the full area SF under the spectrum resembles that of the Debye-Waller factor and that quadrupole splitting decreases drastically, from Δ = 0.7 mm/s to Δ ≈ 0, as the temperature is increased from 220 to 270 K. The mathematical treatment of the spectra and decomposition into three components corresponding to thermal, diffusional and conformational motions enabled us to explain the temperature-dependent changes in the spectra.

  2. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  3. Crystallizing membrane proteins for structure-function studies using lipidic mesophases.

    PubMed

    Caffrey, Martin

    2011-06-01

    The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3½ years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.

  4. Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

    PubMed Central

    Andersen, J N; Elson, A; Lammers, R; Rømer, J; Clausen, J T; Møller, K B; Møller, N P

    2001-01-01

    To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function. PMID:11237862

  5. Molecular dynamics of membrane proteins.

    SciTech Connect

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

    2004-10-01

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

  6. The degree of resistance of erythrocyte membrane cytoskeletal proteins to supra-physiologic concentrations of calcium: an in vitro study.

    PubMed

    Mostafavi, Ebrahim; Nargesi, Arash Aghajani; Ghazizadeh, Zaniar; Larry, Mehrdad; Farahani, Roya Horabad; Morteza, Afsaneh; Esteghamati, Alireza; Vigneron, Claude; Nakhjavani, Manouchehr

    2014-08-01

    Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25-10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R(2) = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.

  7. NMR study of a membrane protein in detergent-free aqueous solution

    PubMed Central

    Zoonens, Manuela; Catoire, Laurent J.; Giusti, Fabrice; Popot, Jean-Luc

    2005-01-01

    One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy–heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP–APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the β-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed. PMID:15956183

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

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

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

  9. Helical Membrane Protein Conformations and their Environment

    PubMed Central

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

    2013-01-01

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

  10. Protein engineering methods applied to membrane protein targets.

    PubMed

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

    2013-02-01

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

  11. Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane1

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2009-03-17

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

  13. Thermodynamic competition between membrane protein oligomeric states

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Haselwandter, Christoph A.

    2016-10-01

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

  14. Electrophysiological characterization of membrane transport proteins.

    PubMed

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

    2013-01-01

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

  15. Maltose neopentyl glycol-3 (MNG-3) analogues for membrane protein study.

    PubMed

    Cho, Kyung Ho; Husri, Mohd; Amin, Anowarul; Gotfryd, Kamil; Lee, Ho Jin; Go, Juyeon; Kim, Jin Woong; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

    2015-05-07

    Detergents are typically used to both extract membrane proteins (MPs) from the lipid bilayers and maintain them in solution. However, MPs encapsulated in detergent micelles are often prone to denaturation and aggregation. Thus, the development of novel agents with enhanced stabilization characteristics is necessary to advance MP research. Maltose neopentyl glycol-3 (MNG-3) has contributed to >10 crystal structures including G-protein coupled receptors. Here, we prepared MNG-3 analogues and characterised their properties using selected MPs. Most MNGs were superior to a conventional detergent, n-dodecyl-β-D-maltopyranoside (DDM), in terms of membrane protein stabilization efficacy. Interestingly, optimal stabilization was achieved with different MNG-3 analogues depending on the target MP. The origin for such detergent specificity could be explained by a novel concept: compatibility between detergent hydrophobicity and MP tendency to denature and aggregate. This set of MNGs represents viable alternatives to currently available detergents for handling MPs, and can be also used as tools to estimate MP sensitivity to denaturation and aggregation.

  16. Quantification of Detergents Complexed with Membrane Proteins

    PubMed Central

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

    2017-01-01

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

  17. Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance

    PubMed Central

    Trahey, Meg; Li, Mavis Jiarong; Kwon, Hyewon; Woodahl, Erica L.; McClary, Wynton D.

    2015-01-01

    Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of BiacoreTM sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody. Advantages and disadvantages of each chip type are considered. PMID:26237675

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

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

    PubMed

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

    2011-11-01

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

  20. Strategies for the purification of membrane proteins.

    PubMed

    Smith, Sinead Marian

    2011-01-01

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

  1. Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

    PubMed Central

    Ruotolo, Roberta; Marchini, Gessica; Ottonello, Simone

    2008-01-01

    Background The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals. Results A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance. Conclusion The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity. PMID:18394190

  2. Proteomic analysis of integral plasma membrane proteins.

    PubMed

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

    2004-04-01

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

  3. Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies.

    PubMed

    Andrews, Daniel A; Xie, Meng; Hughes, Victoria; Wilce, Matthew C; Roujeinikova, Anna

    2013-02-01

    The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This 'solubilization by design' approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.

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

    PubMed

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

    2013-06-04

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

  5. Quantitative ToF-SIMS Studies of Protein Drug Release from Biodegradable Polymer Drug Delivery Membranes

    PubMed Central

    Burns, Sarah A.; Gardella, Joseph A.

    2008-01-01

    Biodegradable polymers are of interest in developing strategies to control protein drug delivery. The protein that was used in this study is Keratinocyte Growth Factor (KGF) which is a protein involved in the re-epithelialization process. The protein is stabilized in the biodegradable polymer matrix during formulation and over the course of polymer degradation with the use of an ionic surfactant Aerosol-OT (AOT) which will encapsulate the protein in an aqueous environment. The release kinetics of the protein from the surface of these materials requires precise timing which is a crucial factor in the efficacy of this drug delivery system. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used in the same capacity to identify the molecular ion peak of the surfactant and polymer and use this to determine surface concentration. In the polymer matrix, the surfactant molecular ion peak was observed in the positive and negative mode at m/z 467 and 421, respectively. These peaks were determined to be [AOT + Na+] and [AOT−Na+]-. These methods are used to identify the surfactant and protein from the polymer matrix and are used to measure the rate of surface accumulation. The second step was to compare this accumulation rate with the release rate of the protein into an aqueous solution during the degradation of the biodegradable film. This rate is compared to that from fluorescence spectroscopy measurements using the protein autofluorescence from that released into aqueous solution. PMID:20016666

  6. Fluorescence spectroscopy of protein oligomerization in membranes.

    PubMed

    Gorbenko, Galyna P

    2011-05-01

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

  7. Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

    PubMed Central

    Corbalan, Natalia; Runti, Giulia; Adler, Conrado; Covaceuszach, Sonia; Ford, Robert C.; Lamba, Doriano; Beis, Konstantinos

    2013-01-01

    SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability. PMID:24078611

  8. Functional and structural study of the dimeric inner membrane protein SbmA.

    PubMed

    Corbalan, Natalia; Runti, Giulia; Adler, Conrado; Covaceuszach, Sonia; Ford, Robert C; Lamba, Doriano; Beis, Konstantinos; Scocchi, Marco; Vincent, Paula A

    2013-12-01

    SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

  9. Computational modeling of membrane proteins

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    Wu, Xiaokun; Han, Min; Ming, Dengming

    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.

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

    NASA Astrophysics Data System (ADS)

    Wu, Xiaokun; Han, Min; Ming, Dengming

    2015-10-01

    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.

  12. Quantitative ToF-SIMS studies of protein drug release from biodegradable polymer drug delivery membranes

    NASA Astrophysics Data System (ADS)

    Burns, Sarah A.; Gardella, Joseph A.

    2008-12-01

    Biodegradable polymers are of interest in developing strategies to control protein drug delivery. The protein that was used in this study is Keratinocyte Growth Factor (KGF) which is a protein involved in the re-epithelialization process. The protein is stabilized in the biodegradable polymer matrix during formulation and over the course of polymer degradation with the use of an ionic surfactant Aerosol-OT (AOT) which will encapsulate the protein in an aqueous environment. The release kinetics of the protein from the surface of these materials requires precise timing which is a crucial factor in the efficacy of this drug delivery system. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used in the same capacity to identify the molecular ion peak of the surfactant and polymer and use this to determine surface concentration. In the polymer matrix, the surfactant molecular ion peak was observed in the positive and negative mode at m/ z 467 and 421, respectively. These peaks were determined to be [AOT + Na +] and [AOT - Na +]. These methods are used to identify the surfactant and protein from the polymer matrix and are used to measure the rate of surface accumulation. The second step was to compare this accumulation rate with the release rate of the protein into an aqueous solution during the degradation of the biodegradable film. This rate is compared to that from fluorescence spectroscopy measurements using the protein autofluorescence from that released into aqueous solution [C.M. Mahoney, J. Yu, A. Fahey, J.A.J. Gardella, SIMS depth profiling of polymer blends with protein based drugs, Appl. Surf. Sci. 252 (2006), 6609-6614.].

  13. Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: What have we learned to date?

    PubMed Central

    McMorran, Lindsay M.; Brockwell, David J.; Radford, Sheena E.

    2014-01-01

    Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins. PMID:24613287

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

    DOEpatents

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

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

  15. Structure and Dynamics of Antifreeze Protein--Model Membrane Interactions: A Combined Spectroscopic and Molecular Dynamics Study.

    PubMed

    Kar, Rajiv K; Mroue, Kamal H; Kumar, Dinesh; Tejo, Bimo A; Bhunia, Anirban

    2016-02-11

    Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.

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

    PubMed Central

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1970-01-01

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

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

    PubMed Central

    Wen, Yi; Dick, Robert A.

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Bhushan, S; Etzel, M R

    2009-04-01

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

  2. Protein permeation through an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    NASA Astrophysics Data System (ADS)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

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

  4. Imaging and structural studies of DNA-protein complexes and membrane ion channels.

    PubMed

    Marini, M; Limongi, T; Falqui, A; Genovese, A; Allione, M; Moretti, M; Lopatin, S; Tirinato, L; Das, G; Torre, B; Giugni, A; Cesca, F; Benfenati, F; Di Fabrizio, E

    2017-02-23

    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 GABAA 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 Å, 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.

  5. Fluctuating hydrodynamics of multicomponent membranes with embedded proteins

    SciTech Connect

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

    2014-08-21

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

  6. Fluctuating hydrodynamics of multicomponent membranes with embedded proteins.

    PubMed

    Camley, Brian A; Brown, Frank L H

    2014-08-21

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

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

    PubMed

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

    2004-11-03

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

  8. Detection of Proteins on Blot Membranes.

    PubMed

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

    2016-11-01

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

  9. Membrane binding of a lipidated N-Ras protein studied in lipid monolayers.

    PubMed

    Bringezu, Frank; Majerowicz, Monika; Wen, Shaoying; Reuther, Guido; Tan, Kui-Thong; Kuhlmann, Jürgen; Waldmann, Herbert; Huster, Daniel

    2007-04-01

    The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding.

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

    PubMed Central

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

    2015-01-01

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

  11. Malate synthase a membrane protein

    SciTech Connect

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

    1987-04-01

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

  12. Internal packing of helical membrane proteins

    PubMed Central

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

    2000-01-01

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

  13. A novel lipoprotein nanoparticle system for membrane proteins

    PubMed Central

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

    2016-01-01

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

  14. Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.

    PubMed

    Shotton, D; Thompson, K; Wofsy, L; Branton, D

    1978-02-01

    We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

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

    PubMed

    Arnold, Thomas; Linke, Dirk

    2007-10-01

    Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.

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

    PubMed

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

    2011-06-01

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

  17. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

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

  18. The human platelet membrane proteome reveals several new potential membrane proteins.

    PubMed

    Moebius, Jan; Zahedi, René Peiman; Lewandrowski, Urs; Berger, Claudia; Walter, Ulrich; Sickmann, Albert

    2005-11-01

    We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.

  19. Subdiffusion of proteins and oligomers on membranes

    NASA Astrophysics Data System (ADS)

    Lepzelter, David; Zaman, Muhammad

    2012-11-01

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

  20. Phylogenetic profiles of all membrane transport proteins

    PubMed Central

    Weiner, January; Kooij, Taco W.A.

    2016-01-01

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

  1. Endogenously Tagged Rab Proteins: A Resource to Study Membrane Trafficking in Drosophila

    PubMed Central

    Dunst, Sebastian; Kazimiers, Tom; von Zadow, Felix; Jambor, Helena; Sagner, Andreas; Brankatschk, Beate; Mahmoud, Ali; Spannl, Stephanie; Tomancak, Pavel; Eaton, Suzanne; Brankatschk, Marko

    2015-01-01

    Summary Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFPMYC at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments. PMID:25942626

  2. Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

    PubMed

    Dunst, Sebastian; Kazimiers, Tom; von Zadow, Felix; Jambor, Helena; Sagner, Andreas; Brankatschk, Beate; Mahmoud, Ali; Spannl, Stephanie; Tomancak, Pavel; Eaton, Suzanne; Brankatschk, Marko

    2015-05-04

    Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.

  3. Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

    PubMed Central

    Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

    2017-01-01

    Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

  4. Toward understanding driving forces in membrane protein folding.

    PubMed

    Hong, Heedeok

    2014-12-15

    α-Helical membrane proteins are largely composed of nonpolar residues that are embedded in the lipid bilayer. An enigma in the folding of membrane proteins is how a polypeptide chain can be condensed into the compact folded state in the environment where the hydrophobic effect cannot strongly drive molecular interactions. Probably other forces such as van der Waals packing, hydrogen bonding, and weakly polar interactions, which are regarded less important in the folding of water-soluble proteins, should emerge. However, it is not clearly understood how those individual forces operate and how they are balanced for stabilizing membrane proteins. Studying this problem is not a trivial task mainly because of the methodological challenges in controlling the reversible folding of membrane proteins in the lipid bilayer. Overcoming the hurdles, meaningful progress has been made in the field in the last few decades. This review will focus on recent studies tackling the problem of driving forces in membrane protein folding.

  5. A comparative study of combined treatment with a collagen membrane and enamel matrix proteins for the regeneration of intraosseous defects.

    PubMed

    Minabe, Masato; Kodama, Toshiro; Kogou, Tatsuji; Takeuchi, Kayo; Fushimi, Hajime; Sugiyama, Takashi; Mitarai, Eiko

    2002-12-01

    Recent clinical study shows that periodontal regeneration therapy using enamel matrix proteins (Emdogain; EMD) is expected to have the same therapeutic effect as guided tissue regeneration (GTR). However, reports on the combined effect of both therapies are limited, and the clinical significance is not definite. In this study, clinical effects were studied by comparing a combination of EMD and GTR using a collagen membrane for intrabony defects with GTR monotherapy and EMD monotherapy. Sixty-one patients with 69 intrabony defects were included. Efficacy of treatment was evaluated at 6 months and 1 year by assessment of reduction of probing depth, probing attachment gain, and radiographic bone gain. There were no statistically significant differences between presurgical soft tissue measurements and defect characteristics for the three treatment groups. The results showed no significant differences in reduction of probing depth, probing attachment gain, or radiographic bone gain between the three treatment groups at both evaluation times. The combination of GTR using a resorbable membrane for intrabony defects and EMD did not enhance the therapeutic effect compared with each monotherapy.

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

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

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

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

    PubMed

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

    2012-11-01

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

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

    PubMed

    Lee, Hunsang; Kim, Hyun

    2014-10-17

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

  9. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-12-11

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  12. Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli.

    PubMed

    Abramson, J; Larsson, G; Byrne, B; Puustinen, A; Garcia-Horsman, A; Iwata, S

    2000-08-01

    Cytochrome bo(3) ubiquinol oxidase has been successfully purified for crystallization. Single crystals of this integral membrane protein diffract X-rays to 3.5 A resolution and belong to the orthorhombic space group C222(1). From the diffraction data, the unit-cell parameters were determined to be a = 91.3, b = 370.3, c = 232.4 A. The crystals have a solvent content of 59% and contain two molecules per asymmetric unit. A search model generated from the structures of cytochrome c oxidase from Paracoccus denitrificans and the extrinsic domain of cytochrome bo(3) ubiquinol oxidase from Escherichia coli was used for molecular-replacement studies, resulting in a solution with sensible molecular packing.

  13. Crystallization of Membrane protein under Microgravity

    NASA Astrophysics Data System (ADS)

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

    2002-01-01

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

  14. Pathogen receptor discovery with a microfluidic human membrane protein array.

    PubMed

    Glick, Yair; Ben-Ari, Ya'ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella; Gerber, Doron

    2016-04-19

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

  15. Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis.

    PubMed Central

    Sato, T; Ohki, M; Yura, T; Ito, K

    1979-01-01

    The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lac- phenotype and temperature-sensitive growth. The mutation could be contransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divE42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a log-phase culture to high temperature (42 degrees C). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used. Images PMID:374381

  16. Membrane Protein Crystallization Using Laser Irradiation

    NASA Astrophysics Data System (ADS)

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

    2004-10-01

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

  17. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  18. A sliding selectivity scale for lipid binding to membrane proteins

    PubMed Central

    Landreh, Michael; Marty, Michael T.; Gault, Joseph; Robinson, Carol V.

    2017-01-01

    Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins that reside in the membrane have co-evolved with their hydrophobic lipid environment which serves as a solvent for proteins with very diverse requirements. As a result, membrane protein-lipid interactions range from completely non-selective to highly discriminating. Mass spectrometry (MS), in combination with X-ray crystallography and molecular dynamics simulations, enables us to monitor how lipids interact with intact membrane protein complexes and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. In this review, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization. PMID:27155089

  19. Studies of the Outer Membrane Proteins of Campylobacter Jejuni for Vaccine Development

    DTIC Science & Technology

    1991-11-26

    serologic tests for diagnosing Campylobacter infection, which could be useful in field settings. 3 4. Identification, purification, and characterization ...response to purified lipopolysaccharide from four Campylobacter strains in 34 adults with inflammatory enteritis in Denver, using an enzyme -linked...Mycobacterium tuberculosis, and M.leprae (66%) and mitochondrial protein p1 precursor of human and Chinese hamster cells (64%), and rubisco subunit binding

  20. Inherently tunable electrostatic assembly of membrane proteins.

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    2009-03-01

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

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

    PubMed

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

    2012-02-25

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

  3. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells.

    PubMed

    Bhatia, Prateek A; Moaddel, Ruin; Wainer, Irving W

    2010-06-15

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.

  4. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

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

    2006-07-01

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

  5. Expression and purification of membrane proteins.

    PubMed

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

    2014-01-01

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

  6. Protein profiles of hatchery egg shell membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    SciTech Connect

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-04-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials.

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

    PubMed

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

    2011-05-04

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

  9. A study of Helicobacter pylori outer-membrane proteins (hom) A and B in Iraq and Turkey.

    PubMed

    Hussein, Nawfal R

    2011-08-01

    Helicobacter pylori outer-membrane proteins (hom), especially the homB gene, have been suggested as a novel virulence factor. However, no study has been conducted in Middle Eastern countries regarding the association between these genes and clinical outcome. Gastric biopsies were obtained from 70 and 64 unselected H. pylori+ patients from Iraq and Turkey, respectively. PCR was performed to study the presence of the homA, homB, and cagA. No association was found between homA or homB and clinical outcomes. When Iraqi and Turkish strains were combined as Middle Eastern samples, cagA positivity was significantly higher in patients with peptic ulcer disease (PUD) than those with non-peptic ulcer disease (NPUD) (p=0.003, OR=3.6, CI=1.5-8.8). Two Iraqi strains showed intermediate, non-homA non-homB, length PCR products. The function of such gene is yet to be determined. In conclusion, hom genes might not be a good indicator for disease prediction in the Middle East. More studies are needed to confirm these results and determine the function of intermediate length hom.

  10. Amyloid Aggregation and Membrane Disruption by Amyloid Proteins

    NASA Astrophysics Data System (ADS)

    Ramamoorthy, Ayyalusamy

    2013-03-01

    Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.

  11. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey V.; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F.

    2016-01-01

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins. PMID:26908053

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

    PubMed

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

    2012-04-01

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

  13. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

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

    PubMed

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

    2000-09-01

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

  15. β-Barrel membrane protein assembly by the Bam complex.

    PubMed

    Hagan, Christine L; Silhavy, Thomas J; Kahne, Daniel

    2011-01-01

    β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

  16. Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

    PubMed

    Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

    2014-08-25

    When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes.

  17. Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins

    PubMed Central

    Beebe, Emily T.; Makino, Shin-ichi; Nozawa, Akira; Matsubara, Yuko; Frederick, Ronnie O.; Primm, John G.; Goren, Michael A.; Fox, Brian G.

    2010-01-01

    The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2 mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins. PMID:20637905

  18. Ponticulin is an atypical membrane protein

    PubMed Central

    1994-01-01

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

  19. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

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

    2012-02-01

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

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

    PubMed

    Mellgren, Ronald L

    2008-04-24

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

  1. Fully Quantified Spectral Imaging Reveals in Vivo Membrane Protein Interactions

    PubMed Central

    King, Christopher; Stoneman, Michael; Raicu, Valerica; Hristova, Kalina

    2016-01-01

    Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling. PMID:26787445

  2. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  3. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  4. Production of okara and soy protein concentrates using membrane technology.

    PubMed

    Vishwanathan, K H; Govindaraju, K; Singh, Vasudeva; Subramanian, R

    2011-01-01

    Microfiltration (MF) membranes with pore sizes of 200 and 450 nm and ultrafiltration (UF) membranes with molecular weight cut off of 50, 100, and 500 kDa were assessed for their ability to eliminate nonprotein substances from okara protein extract in a laboratory cross-flow membrane system. Both MF and UF improved the protein content of okara extract to a similar extent from approximately 68% to approximately 81% owing to the presence of protein in the feed leading to the formation of dynamic layer controlling the performance rather than the actual pore size of membranes. Although normalized flux in MF-450 (117 LMH/MPa) was close to UF-500 (118 LMH/MPa), the latter was selected based on higher average flux (47 LMH) offering the advantage of reduced processing time. Membrane processing of soy extract improved the protein content from 62% to 85% much closer to the target value. However, the final protein content in okara (approximately 80%) did not reach the target value (90%) owing to the greater presence of soluble fibers that were retained by the membrane. Solubility curve of membrane okara protein concentrate (MOPC) showed lower solubility than soy protein concentrate and a commercial isolate in the entire pH range. However, water absorption and fat-binding capacities of MOPC were either superior or comparable while emulsifying properties were in accordance with its solubility. The results of this study showed that okara protein concentrate (80%) could be produced using membrane technology without loss of any true proteins, thus offering value addition to okara, hitherto underutilized. Practical Application: Okara, a byproduct obtained during processing soybean for soymilk, is either underutilized or unutilized in spite of the fact that its protein quality is as good as that of soy milk and tofu. Membrane-processed protein products have been shown to possess superior functional properties compared to conventionally produced protein products. However, the

  5. Intrinsically disordered proteins drive membrane curvature

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  6. Intrinsically disordered proteins drive membrane curvature

    PubMed Central

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

    2015-01-01

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

  7. Intrinsically disordered proteins drive membrane curvature.

    PubMed

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

    2015-07-24

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

  8. Membrane–drug interactions studied using model membrane systems

    PubMed Central

    Knobloch, Jacqueline; Suhendro, Daniel K.; Zieleniecki, Julius L.; Shapter, Joseph G.; Köper, Ingo

    2015-01-01

    The direct interaction of drugs with the cell membrane is often neglected when drug effects are studied. Systematic investigations are hindered by the complexity of the natural membrane and model membrane systems can offer a useful alternative. Here some examples are reviewed of how model membrane architectures including vesicles, Langmuir monolayers and solid supported membranes can be used to investigate the effects of drug molecules on the membrane structure, and how these interactions can translate into effects on embedded membrane proteins. PMID:26586998

  9. Protein transfer to membranes upon shape deformation

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

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

  10. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases.

  11. Protein separation using an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

    Jou, Ining; Melnikov, Dmitriy; Gracheva, Maria

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

  12. Protein aggregation in a membrane environment.

    PubMed

    Gorbenko, Galyna; Trusova, Valeriya

    2011-01-01

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

  13. Breaking the barriers in membrane protein crystallography.

    PubMed

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

    2013-03-01

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

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

    PubMed Central

    Milovanovic, Dragomir; Jahn, Reinhard

    2015-01-01

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

  15. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

    SciTech Connect

    Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y.L.; Doniach, Sebastian; Lesley, Scott A.

    2009-10-21

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

  16. The membrane-water interface region of membrane proteins: structural bias and the anti-snorkeling effect.

    PubMed

    Liang, Jie; Adamian, Larisa; Jackups, Ronald

    2005-07-01

    Membrane proteins have important roles in many cellular processes. Computational analysis of their sequences and structures has provided much insight into the organizing principles of transmembrane helices. In a recent study, the membrane-water interface region was examined in detail for the first time. The results have revealed that this interface region has an important role in constraining protein secondary structure. This study raises new questions and opens up new directions for studying membrane proteins.

  17. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices.

  18. Vaccinia virus virion membrane biogenesis protein A11 associates with viral membranes in a manner that requires the expression of another membrane biogenesis protein, A6.

    PubMed

    Wu, Xiang; Meng, Xiangzhi; Yan, Bo; Rose, Lloyd; Deng, Junpeng; Xiang, Yan

    2012-10-01

    A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

  19. Membrane Protein Structure, Function and Dynamics: A Perspective from Experiments and Theory

    PubMed Central

    Cournia, Zoe; Allen, Toby W.; Andricioaei, Ioan; Antonny, Bruno; Baum, Daniel; Brannigan, Grace; Buchete, Nicolae-Viorel; Deckman, Jason T.; Delemotte, Lucie; del Val, Coral; Friedman, Ran; Gkeka, Paraskevi; Hege, Hans-Christian; Hénin, Jérôme; Kasimova, Marina A.; Kolocouris, Antonios; Klein, Michael L.; Khalid, Syma; Lemieux, M. Joanne; Lindow, Norbert; Roy, Mahua; Selent, Jana; Tarek, Mounir; Tofoleanu, Florentina; Vanni, Stefano; Urban, Sinisa; Wales, David J.; Smith, Jeremy C.; Bondar, Ana-Nicoleta

    2015-01-01

    Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes, receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function and dynamics of membrane proteins. PMID:26063070

  20. Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory

    DOE PAGES

    Cournia, Zoe; Allen, Toby W.; Andricioaei, Ioan; ...

    2015-06-11

    It is fundamental for the flourishing biological cells that membrane proteins mediate the process. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. Here, we present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

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

    NASA Astrophysics Data System (ADS)

    Sun, Junfen; Wu, Lishun

    2014-07-01

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

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

    PubMed

    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).

  3. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

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

    2014-01-01

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

  4. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    -bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA. PMID:26912608

  5. Curvature-mediated interactions between membrane proteins.

    PubMed Central

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

    1998-01-01

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

  6. Exceptional overproduction of a functional human membrane protein.

    PubMed

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

    2007-11-01

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

  7. Predictive energy landscapes for folding membrane protein assemblies

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  8. Model-building codes for membrane proteins.

    SciTech Connect

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

    2005-01-01

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

  9. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  10. Predicting membrane protein types with bragging learner.

    PubMed

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

    2008-01-01

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

  11. Membranes: a meeting point for lipids, proteins and therapies

    PubMed Central

    Escribá, Pablo V; González-Ros, José M; Goñi, Félix M; Kinnunen, Paavo K J; Vigh, Lászlo; Sánchez-Magraner, Lissete; Fernández, Asia M; Busquets, Xavier; Horváth, Ibolya; Barceló-Coblijn, Gwendolyn

    2008-01-01

    Abstract Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy. PMID:18266954

  12. Membranes: a meeting point for lipids, proteins and therapies.

    PubMed

    Escribá, Pablo V; González-Ros, José M; Goñi, Félix M; Kinnunen, Paavo K J; Vigh, Lászlo; Sánchez-Magraner, Lissete; Fernández, Asia M; Busquets, Xavier; Horváth, Ibolya; Barceló-Coblijn, Gwendolyn

    2008-06-01

    Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

  13. Subcellular localization of mammalian type II membrane proteins.

    PubMed

    Aturaliya, Rajith N; Fink, J Lynn; Davis, Melissa J; Teasdale, Melvena S; Hanson, Kelly A; Miranda, Kevin C; Forrest, Alistair R R; Grimmond, Sean M; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-05-01

    Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

  14. Proteomics characterization of abundant Golgi membrane proteins.

    PubMed

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

    2001-02-16

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

  15. Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

    PubMed

    Ashok, Yashwanth; Jaakola, Veli-Pekka

    2016-01-01

    Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

  16. Major intrinsic proteins in biomimetic membranes.

    PubMed

    Nielsen, Claus Hélix

    2010-01-01

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

  17. Simple model of membrane proteins including solvent.

    PubMed

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

    2006-05-14

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

  18. Atomic force microscopy and spectroscopy of native membrane proteins.

    PubMed

    Müller, Daniel J; Engel, Andreas

    2007-01-01

    Membrane proteins comprise 30% of the proteome of higher organisms. They mediate energy conversion, signal transduction, solute transport and secretion. Their native environment is a bilayer in a physiological buffer solution, hence their structure and function are preferably assessed in this environment. The surface structure of single membrane proteins can be determined in buffer solutions by atomic force microscopy (AFM) at a lateral resolution of less than 1 nm and a vertical resolution of 0.1-0.2 nm. Moreover, single proteins can be directly addressed, stuck to the AFM stylus and subsequently unfolded, revealing the molecular interactions of the protein studied. The examples discussed here illustrate the power of AFM in the structural analysis of membrane proteins in a native environment.

  19. Isothermal titration calorimetry of membrane proteins - progress and challenges.

    PubMed

    Rajarathnam, Krishna; Rösgen, Jörg

    2014-01-01

    Integral membrane proteins, including G protein-coupled receptors (GPCR) and ion channels, mediate diverse biological functions that are crucial to all aspects of life. The knowledge of the molecular mechanisms, and in particular, the thermodynamic basis of the binding interactions of the extracellular ligands and intracellular effector proteins is essential to understand the workings of these remarkable nanomachines. In this review, we describe how isothermal titration calorimetry (ITC) can be effectively used to gain valuable insights into the thermodynamic signatures (enthalpy, entropy, affinity, and stoichiometry), which would be most useful for drug discovery studies, considering that more than 30% of the current drugs target membrane proteins. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

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

    PubMed

    Okamoto, Yukihiro; Tsujimoto, Yusuke; Umakoshi, Hiroshi

    2016-03-01

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

  1. Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid–protein interaction

    PubMed Central

    Mangialavori, Irene C.; Corradi, Gerardo; Rinaldi, Débora E.; delaFuente, María Candelaria; Adamo, Hugo P.; Rossi, Juan Pablo F. C.

    2014-01-01

    The autoinhibition/activation of the PMCA (plasma membrane Ca2+ -ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid–protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid–protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA. PMID:22214540

  2. Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction.

    PubMed

    Mangialavori, Irene C; Corradi, Gerardo; Rinaldi, Débora E; de la Fuente, María Candelaria; Adamo, Hugo P; Rossi, Juan Pablo F C

    2012-04-01

    The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.

  3. Directional interactions and cooperativity between mechanosensitive membrane proteins

    NASA Astrophysics Data System (ADS)

    Haselwandter, Christoph A.; Phillips, Rob

    2013-03-01

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

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

    PubMed Central

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

    2002-01-01

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

  5. Folding studies of Purified LamB Protein, the Maltoporin from the Escherichia coli Outer Membrane: Trimer Dissociation can be separated from Unfolding

    PubMed Central

    Baldwin, Valerie; Bhatia, Mandeep; Luckey, Mary

    2011-01-01

    The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure. PMID:21640073

  6. Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: trimer dissociation can be separated from unfolding.

    PubMed

    Baldwin, Valerie; Bhatia, Mandeep; Luckey, Mary

    2011-09-01

    The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.

  7. The cost of living in the membrane: a case study of hydrophobic mismatch for the multi-segment protein LeuT.

    PubMed

    Mondal, Sayan; Khelashvili, George; Shi, Lei; Weinstein, Harel

    2013-04-01

    Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the

  8. Membrane Proteins in Four Acts: Function Precedes Structure Determination

    PubMed Central

    Cramer, W. A.; Zakharov, S. D.; Hasan, S. Saif; Zhang, H.; Baniulis, D.; Zhalnina, M. V.; Soriano, G. M.; Sharma, O.; Rochet, J. C.; Ryan, C.; Whitelegge., J.; Kurisu, G.; Yamashita, E.

    2011-01-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/ proton translocation). (1) Crystal structures of the eight subunit heterooligomeric trans-membrane dimeric cytochrome b6f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of seventeen monotopic and polytopic hetero-subunits. (II) β-barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins (1). A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained (2). A crystal structure of the N-terminal translocation domain of colicin E3

  9. The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

    PubMed

    Habicht, K-L; Frazier, C; Singh, N; Shimmo, R; Wainer, I W; Moaddel, R

    2013-01-01

    BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

  10. Molecular dynamics simulations of a membrane protein/amphipol complex.

    PubMed

    Perlmutter, Jason D; Popot, Jean-Luc; Sachs, Jonathan N

    2014-10-01

    Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

  11. Self diffusion of interacting membrane proteins.

    PubMed Central

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

    1989-01-01

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

  12. The heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans: relationship to OmpA proteins.

    PubMed Central

    Wilson, M E

    1991-01-01

    The outer membrane of Actinobacillus actinomycetemcomitans contains a 29-kDa protein which exhibits heat modifiability on sodium dodecyl sulfate-polyacrylamide gels and represents a major target for immunoglobulin G antibody in sera of periodontitis patients colonized by this organism. In the present study, the N-terminal amino acid sequence of the 29-kDa outer membrane protein was determined and compared with reported sequences for other known proteins. The heat-modifiable outer membrane protein of A. actinomycetemcomitans was found to exhibit significant N-terminal homology with the OmpA proteins of other gram-negative bacteria. Moreover, this protein reacted with antiserum raised against the purified OmpA protein of Escherichia coli K-12. Whether the heat-modifiable OMP of A. actinomycetemcomitans also shares functional properties of OmpA proteins, particularly with respect to bacteriophage receptor activity, is presently under investigation. Images PMID:2050416

  13. Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

    PubMed Central

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

    2014-01-01

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

  14. Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis.

    PubMed

    Peker, Selen; Akar, Nejat; Demiralp, Duygu Ozel

    2012-03-01

    Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-β, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.

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

    PubMed Central

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

    2015-01-01

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

  16. Atomic-level description of protein-lipid interactions using an accelerated membrane model.

    PubMed

    Baylon, Javier L; Vermaas, Josh V; Muller, Melanie P; Arcario, Mark J; Pogorelov, Taras V; Tajkhorshid, Emad

    2016-07-01

    Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov.

  17. Sculpting membranes: a mechanism of curvature generation by proteins

    NASA Astrophysics Data System (ADS)

    Campelo, Felix

    2010-03-01

    A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Crescent shaped N-BAR domains containing amphipathic helices can induce membrane curvature by two mechanisms: the scaffolding mechanism due to the very shape of the BAR dimer, and the hydrophobic insertion mechanism by which small shallow inclusions penetrate the membrane matrix and act as a wedge changing the local membrane curvature. We will focus on this latter mechanism, and study it from a quantitative point of view. We use an elastic model of the lipid bilayer, taking into account the internal strains and stresses generated by the presence of an inclusion. We show that large membrane curvatures found in in vitro experiments can be ascribed to this mechanism, and that shallow insertions are more powerful curvature generators than lipids.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-07-30

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Landreh, Michael; Robinson, Carol V

    2015-01-01

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

  2. Protein–protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins

    PubMed Central

    Kleanthous, Colin; Rassam, Patrice; Baumann, Christoph G

    2015-01-01

    It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises. PMID:26629934

  3. Novel silk protein barrier membranes for guided bone regeneration.

    PubMed

    Smeets, Ralf; Knabe, Christine; Kolk, Andreas; Rheinnecker, Michael; Gröbe, Alexander; Heiland, Max; Zehbe, Rolf; Sachse, Manuela; Große-Siestrup, Christian; Wöltje, Michael; Hanken, Henning

    2016-10-12

    This study assesses the biocompatibility of novel silk protein membranes with and without modification, and evaluates their effect on facilitating bone formation and defect repair in guided bone regeneration. Two calvarian bone defects 12 mm in diameter were created in each of a total of 38 rabbits. Four different types of membranes, (silk-, hydroxyapatite-modified silk-, β-TCP-modified silk- and commonly clinically used collagen-membranes) were implanted to cover one of the two defects in each animal. Histologic analysis did not show any adverse tissue reactions in any of the defect sites indicating good biocompatibility of all silk protein membranes. Histomorphometric and histologic evaluation revealed that collagen and β-TCP modified silk membranes supported bone formation (collagen: bone area fraction p = 0.025; significant; β-TCP modified silk membranes bone area fraction: p = 0.24, not significant), guided bone regeneration and defect bridging. The bone, which had formed in defects covered by β-TCP modified silk membranes, displayed a more advanced stage of bone tissue maturation with restoration of the original calvarial bone microarchitecture when compared to the bone which had formed in defects, for which any of the other test membranes were used. Micro-CT analysis did not reveal any differences in the amount of bone formation between defects with and without membranes. In contrast to the collagen membranes, β-TCP modified silk membranes were visible in all cases and may therefore be advantageous for further supporting bone formation beyond 10 weeks and preventing soft tissue ingrowth from the periphery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

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

    PubMed

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

    2017-02-06

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

  5. Molecular mechanisms of protein and lipid targeting to ciliary membranes

    PubMed Central

    Emmer, Brian T.; Maric, Danijela; Engman, David M.

    2010-01-01

    Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle. PMID:20145001

  6. Novel Xylene-linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation

    PubMed Central

    Cho, Kyung Ho; Du, Yang; Scull, Nicola J; Hariharan, Parameswaran; Gotfryd, Kamil; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Kobilka, Brian K; Chae, Pil Seok

    2015-01-01

    Membrane proteins are key functional players in biological systems. These bio-macromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, we developed a novel class of maltoside-bearing amphiphiles, with a xylene linker in the central region, designated xylene-linked maltoside amphiphiles (XMAs). When these novel agents were evaluated with a number of membrane proteins, we found that XMA-4 and XMA-5 have particularly favorable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study. PMID:26013293

  7. Steady-state compartmentalization of lipid membranes by active proteins.

    PubMed Central

    Sabra, M C; Mouritsen, O G

    1998-01-01

    Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation. PMID:9533687

  8. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

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

    2009-01-01

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

  9. Light- and pH-dependent conformational changes in protein structure induce strong bending of purple membranes--active membranes studied by cryo-SEM.

    PubMed

    Rhinow, Daniel; Hampp, Norbert A

    2008-10-16

    Bacteriorhodopsin (BR) undergoes a conformational change during the photocycle and the proton transport through the membrane. For the first time, we could demonstrate by direct imaging of freely suspended native purple membranes (PMs) that the flat disk-like shape of PMs changes dramatically as soon as most of the BRs are in a state characterized by a deprotonated Schiff base. Light-induced shape changes are easily observed with mutated BRs of the BR-D96N type, i.e., all variants which show an increased M 2 lifetime. On the other hand, large-scale shape changes are induced by pH changes with PM containing mutated BRs of the BR-D85T type, where Asp85 is replaced for a neutral amino acid. In such PMs, all BRs are titrated simultaneously and the resulting shape of the membranes depends on the initial shape only. As the majority of PMs in the "flat" state are more or less round disks, the bent membranes often comprise bowl-like and tube-like bent structures. The method presented here enables one to derive size changes of membrane-embedded BRs on the single molecule level from "macroscopic", easily accessible data like the curvature radii observed in cryo-SEM. The potential of BR as a pH-controlled and/or light-controlled microscaled biological actuator needs further consideration.

  10. Pathogen receptor discovery with a microfluidic human membrane protein array

    PubMed Central

    Glick, Yair; Ben-Ari, Ya’ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella

    2016-01-01

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein–pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism. PMID:27044079

  11. Vesicle permeabilization by purified soluble oligomers of prion protein: a comparative study of the interaction of oligomers and monomers with lipid membranes.

    PubMed

    Chich, J-F; Chapuis, C; Henry, C; Vidic, J; Rezaei, H; Noinville, S

    2010-04-09

    The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

  12. Studies on gonococcus infection. XVIII. 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane.

    PubMed Central

    Swanson, J

    1979-01-01

    The major outer membrane proteins from 10 gonococcal strains were examined after 125I-labeling of the proteins as single bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These 125I-proteins were then treated with either trypsin or alpha-chymotrypsin, and the resultant 125I-peptides were visualized by autoradiography after two-dimensional electrophoretic and chromatographic separation on thin-layer cellulose sheets. Several 125I-peptides were present in all the major outer membrane proteins examined. The presence and absence of additional 125I-peptides segregated the major proteins into two pattern groups. One group consisted of major outer membranes with molecular weights of 34,000 or 33,000; major proteins with molecular weights of 32,000 constituted the other group. Two beta-lactamase-producing gonococcal isolates were examined. Their major outer membrane proteins were identical in apparent molecular weights and alpha-chymotryptic 125I-peptide fingerprints; these proteins contained 125I-peptides not found in other gonococcal major proteins. No 125I-peptide differences were found among the major outer membrane proteins of strain F62 gonococci that exhibited differences in piliation and/or colony opacity characteristics. Images PMID:110681

  13. Curvature Sorting of Peripheral Proteins on Solid-Supported Wavy Membranes

    PubMed Central

    Hsieh, Wan-Ting; Hsu, Chih-Jung; Capraro, Benjamin R.; Wu, Tingting; Chen, Chi-Mon; Yang, Shu; Baumgart, Tobias

    2013-01-01

    Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures, and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate imposing a range of curvatures onto an adhering lipid bilayer membrane. We examined the curvature sorting of several peripheral proteins binding to the wavy membrane and observed them to partition into distinct regions of curvature. Furthermore, single molecule imaging experiments suggested that curvature sensing of proteins on low-curvature substrates requires cooperative interactions. PMID:22881196

  14. Integral membrane protein interaction with Triton cytoskeletons of erythrocytes.

    PubMed

    Sheetz, M P

    1979-10-19

    The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.

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

    PubMed

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

    2011-04-06

    Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.

  16. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels.

    PubMed

    Itel, Fabian; Al-Samir, Samer; Öberg, Fredrik; Chami, Mohamed; Kumar, Manish; Supuran, Claudiu T; Deen, Peter M T; Meier, Wolfgang; Hedfalk, Kristina; Gros, Gerolf; Endeward, Volker

    2012-12-01

    Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO(2), using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.

  17. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Simunovic, Mijo; Voth, Gregory A.

    2015-05-01

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

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

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

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

  20. Detergent interaction with tethered bilayer lipid membranes for protein reconstitution

    NASA Astrophysics Data System (ADS)

    Broccio, Matteo; Zan Goh, Haw; Loesche, Mathias

    2009-03-01

    Tethered bilayer lipid membranes (tBLMs) are self-assembled biomimetic structures in which the membrane is separated from a solid substrate by a nm-thick hydrated submembrane space. These model systems are being used in binding studies of peripheral proteins and exotoxins. Here we aim at their application for the reconstitution of water-insoluble integral membrane proteins. As an alternative to fusion of preformed proteoliposomes we study the direct reconstitution of such proteins for applications in biosensing and pharmaceutical screening. For reconstitution, highly insulating tBLMs (R˜10^5-10^6 φ) were temporarily incubated with a detergent to screen for conditions that keep the detergent-saturated membranestable and ready to incorporate detergent-solubilized proteins. We assess the electrical characteristics, i.e. specific resistance and capacitance, by means of electrochemical impedance spectroscopy (EIS) under timed incubation with decylmaltoside and dodecylmaltoside detergents in a regime around their critical micelle concentration, 1.8 mM and 0.17 mM respectively and demonstrate the restoration of the tBLM upon detergent removal. Thereby a range of concentration and incubation times was identified, that represents optimal conditions for the subsequent membrane protein reconstitution.

  1. bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles.

    PubMed

    Zheng, Hui; Lee, Sungsoo; Llaguno, Marc C; Jiang, Qiu-Xing

    2016-01-01

    Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo-electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2-20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2-0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid-protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and

  2. Proteomic characterization of integral membrane proteins using thermostatted liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Moore, Sarah M; Wu, Christine C

    2012-01-01

    Due to the hydrophobicity and localization of integral membrane proteins, they are difficult to study using conventional biochemical methods that are compatible with proteomic analyses. This chapter describes the coupling of multiple crucial steps that lead to the optimized shotgun proteomic analysis of integral membrane proteins while maintaining empirical topology information. Namely, a membrane shaving method is utilized to separate protease accessible peptides from membrane embedded peptides and elevated temperatures during chromatographic separation is utilized to augment the recovery of hydrophobic peptides for in-line analysis using tandem mass spectrometry. This combination of steps facilitates increased identification of membrane proteins while also maintaining information regarding protein topology.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  6. A role for the membrane Golgi protein Ema in autophagy.

    PubMed

    Kim, Sungsu; DiAntonio, Aaron

    2012-08-01

    Autophagy is a cellular homeostatic response that involves degradation of self-components by the double-membraned autophagosome. The biogenesis of autophagosomes has been well described, but the ensuing processes after autophagosome formation are not clear. In our recent study, we proposed a model in which the Golgi complex contributes to the growth of autophagic structures, and that the Drosophila melanogaster membrane protein Ema promotes this process. In fat body cells of the D. melanogaster ema mutant, the recruitment of the Golgi complex protein Lava lamp (Lva) to autophagic structures is impaired and autophagic structures are very small. In addition, in the ema mutant autophagic turnover of SQSTM1/p62 and mitophagy are impaired. Our study not only identifies a role for Ema in autophagy, but also supports the hypothesis that the Golgi complex may be a potential membrane source for the biogenesis and development of autophagic structures.

  7. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  8. Membrane curvature and its generation by BAR proteins

    PubMed Central

    Mim, Carsten; Unger, Vinzenz M

    2012-01-01

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

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

    PubMed Central

    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. PMID:24381579

  10. mRNA-programmed translation pauses in the targeting of E. coli membrane proteins.

    PubMed

    Fluman, Nir; Navon, Sivan; Bibi, Eitan; Pilpel, Yitzhak

    2014-08-18

    In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous signal recognition particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously, however, the Alu domain is lacking in most eubacteria. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine-Dalgarno-like elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control the translation speed.

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

    PubMed

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

    1984-06-01

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

  12. Identification of membrane proteins by tandem mass spectrometry of protein ions.

    PubMed

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

    2007-09-04

    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 alpha-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 alpha-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 alpha-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.

  13. The size and detergent binding of membrane proteins.

    PubMed

    Clarke, S

    1975-07-25

    Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.

  14. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes.

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

    PubMed

    Goode, Robert J A; Simpson, Richard J

    2009-01-01

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

  16. Membrane proteins in four acts: function precedes structure determination.

    PubMed

    Cramer, W A; Zakharov, S D; Saif Hasan, S; Zhang, H; Baniulis, D; Zhalnina, M V; Soriano, G M; Sharma, O; Rochet, J C; Ryan, C; Whitelegge, J; Kurisu, G; Yamashita, E

    2011-12-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to

  17. Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families.

    PubMed

    Patel, Prianka V; Gianoulis, Tara A; Bjornson, Robert D; Yip, Kevin Y; Engelman, Donald M; Gerstein, Mark B

    2010-07-01

    Recent metagenomics studies have begun to sample the genomic diversity among disparate habitats and relate this variation to features of the environment. Membrane proteins are an intuitive, but thus far overlooked, choice in this type of analysis as they directly interact with the environment, receiving signals from the outside and transporting nutrients. Using global ocean sampling (GOS) data, we found nearly approximately 900,000 membrane proteins in large-scale metagenomic sequence, approximately a fifth of which are completely novel, suggesting a large space of hitherto unexplored protein diversity. Using GPS coordinates for the GOS sites, we extracted additional environmental features via interpolation from the World Ocean Database, the National Center for Ecological Analysis and Synthesis, and empirical models of dust occurrence. This allowed us to study membrane protein variation in terms of natural features, such as phosphate and nitrate concentrations, and also in terms of human impacts, such as pollution and climate change. We show that there is widespread variation in membrane protein content across marine sites, which is correlated with changes in both oceanographic variables and human factors. Furthermore, using these data, we developed an approach, protein families and environment features network (PEN), to quantify and visualize the correlations. PEN identifies small groups of covarying environmental features and membrane protein families, which we call "bimodules." Using this approach, we find that the affinity of phosphate transporters is related to the concentration of phosphate and that the occurrence of iron transporters is connected to the amount of shipping, pollution, and iron-containing dust.

  18. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

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

  19. Can proteins be intrinsically disordered inside a membrane?

    PubMed Central

    Kjaergaard, Magnus

    2015-01-01

    Intrinsically disorder has evolved in many soluble proteins because it confers a unique set of functional advantages. In contrast, the functions of membrane proteins are largely understood in terms of well-defined structures. This raises the question: Why would the evolutionary pressures that select for disorder leave membrane proteins untouched. In this hypothesis piece, I argue that intrinsic disorder may exist in membrane embedded proteins, but that it will take a different form due to the different environment. Disordered membrane proteins are thus likely to have fully formed secondary structure, but little tertiary structure. Furthermore, the sequence signature for disorder in membrane proteins is likely to be reversed; so disordered proteins are more hydrophobic than their folded counterparts. At present it is impossible to tell how common this type of disordered membrane protein is.

  20. High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

    PubMed

    Walian, Peter J; Allen, Simon; Shatsky, Maxim; Zeng, Lucy; Szakal, Evelin D; Liu, Haichuan; Hall, Steven C; Fisher, Susan J; Lam, Bonita R; Singer, Mary E; Geller, Jil T; Brenner, Steven E; Chandonia, John-Marc; Hazen, Terry C; Witkowska, H Ewa; Biggin, Mark D; Jap, Bing K

    2012-12-07

    Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

  1. Cholesterol and the interaction of proteins with membrane domains.

    PubMed

    Epand, Richard M

    2006-07-01

    Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in

  2. Durable vesicles for reconstitution of membrane proteins in biotechnology

    PubMed Central

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

    2017-01-01

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

  3. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli*

    PubMed Central

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H.; Pessi, Gabriella; Eberl, Leo; Robinson, John A.

    2016-01-01

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  4. G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

    PubMed

    Álvarez, Rafael; López, David J; Casas, Jesús; Lladó, Victoria; Higuera, Mónica; Nagy, Tünde; Barceló, Miquel; Busquets, Xavier; Escribá, Pablo V

    2015-11-01

    G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

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

    PubMed

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

    2015-03-01

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

  6. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

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

    PubMed Central

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

    2011-01-01

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

  8. Label-free study of the function of ion channel protein on a microfluidic optical sensor integrated with artificial cell membrane.

    PubMed

    Li, Zhen; Tang, Yanyan; Zhang, Ling; Wu, Jianmin

    2014-01-21

    A label-free optical sensor was constructed by integrating pH sensing material and supported phospholipid bilayers (SPBs) in a microfluidic chip. The pH sensing material was composed of a double layer structure consisting of chitosan hydrogel and electrochemically etched porous silicon. The pH change in the microchip could induce a reversible swelling of the chitosan hydrogel layer and consequently caused a shift in effective optical thickness (EOT) of the double layer, which could be observed by Fourier transformed reflectometric interference spectroscopy (FT-RIS). After phospholipid bilayers (PLBs) were self-assembled on the sensing layer, the EOT almost remained constant during the cycling of pH from 7.4 to 6.2, indicating the blockage of H(+) translocation by the PLBs. For studying the behavior of ion channel protein, gramicidin A, a typical ion channel protein, was inserted in the SPBs for mimicking the ion transportation function of cell membrane. Due to the H(+) transportation capability of gramicidin A, the optical response to pH change could partially recover. In the presence of Ca(2+), the pore of the ion channel protein was blocked, causing a significant decrease in the EOT response upon pH change. The bio-functionalized microfluidic sensor fabricated in this work will provide a reliable platform for studying the function of ion channel protein, which is an important class of drug targets.

  9. Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

    PubMed Central

    Devauges, Viviane; Marquer, Catherine; Lécart, Sandrine; Cossec, Jack-Christophe; Potier, Marie-Claude; Fort, Emmanuel; Suhling, Klaus; Lévêque-Fort, Sandrine

    2012-01-01

    Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer’s disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions. PMID:22973448

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

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

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

  11. More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes.

    PubMed

    Ros, Uris; García-Sáez, Ana J

    2015-06-01

    Pore-forming proteins (PFPs) punch holes in their target cell membrane to alter their permeability. Permeabilization of lipid membranes by PFPs has received special attention to study the basic molecular mechanisms of protein insertion into membranes and the development of biotechnological tools. PFPs act through a general multi-step mechanism that involves (i) membrane partitioning, (ii) insertion into the hydrophobic core of the bilayer, (iii) oligomerization, and (iv) pore formation. Interestingly, PFPs and membranes show a dynamic interplay. As PFPs are usually produced as soluble proteins, they require a large conformational change for membrane insertion. Moreover, membrane structure is modified upon PFPs insertion. In this context, the toroidal pore model has been proposed to describe a pore architecture in which not only protein molecules but also lipids are directly involved in the structure. Here, we discuss how PFPs and lipids cooperate and remodel each other to achieve pore formation, and explore new evidences of protein-lipid pore structures.

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

    PubMed

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

    2014-01-01

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

  13. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  14. Effects of L-arginine on solubilization and purification of plant membrane proteins.

    PubMed

    Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

    2011-11-01

    Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins.

  15. Continuum electromechanical modeling of protein-membrane interactions.

    PubMed

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

    2010-10-01

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

  16. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    PubMed

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  17. Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

    PubMed Central

    Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

    2014-01-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

  18. Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings

    PubMed Central

    Bibow, Stefan; Carneiro, Marta G; Sabo, T Michael; Schwiegk, Claudia; Becker, Stefan; Riek, Roland; Lee, Donghan

    2014-01-01

    Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment. PMID:24752984

  19. Cell-free methods to produce structurally intact mammalian membrane proteins

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1–1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  20. Probing protein-lipid interactions by FRET between membrane fluorophores

    NASA Astrophysics Data System (ADS)

    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.

  1. Evolution of a Membrane Protein Regulon in Saccharomyces

    PubMed Central

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

    2012-01-01

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

  2. Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.

    PubMed Central

    Poruchynsky, M S; Atkinson, P H

    1991-01-01

    Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process. Images PMID:1651404

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

    PubMed

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

    2015-04-01

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

  4. Physiological Response to Membrane Protein Overexpression in E. coli*

    PubMed Central

    Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K.; Luff, Jon D.; Boël, Grégory; Gauthier, Nils; Handelman, Samuel K.; Ades, Sarah E.; Hunt, John F.

    2011-01-01

    Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially

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

    PubMed Central

    1991-01-01

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

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

    PubMed

    Jeffery, Constance J

    2016-02-02

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

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

    DOE PAGES

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; ...

    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. Wemore » 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. In conclusion, 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.« less

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

    SciTech Connect

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjaer; 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. In conclusion, 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.

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

    PubMed

    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.

  10. Understanding leaf membrane protein extraction to develop a food-grade process.

    PubMed

    Tamayo Tenorio, Angelica; Boom, Remko M; van der Goot, Atze Jan

    2017-02-15

    Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics. Therefore, this study analysed proteomic extraction methods for membrane proteins as an inspiration for a food-grade alternative process. Sugar beet leaves were extracted with two proteomic protocols: solvent extraction and Triton X-114 phase partitioning method. Extraction steps contributed to protein purity and/or to selective fractionation, enabling the purification of specific proteins. It was observed that membrane proteins distributed among different solvents, buffers and solutions used due to their physicochemical heterogeneity. This heterogeneity does not allow a total membrane protein extraction by a unique method or even combinations of processing steps, but it enables the creation of different fractions with different physicochemical properties useful for food applications.

  11. Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes

    PubMed Central

    1981-01-01

    Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA- dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the

  12. Therapeutic design of peptide modulators of protein-protein interactions in membranes.

    PubMed

    Stone, Tracy A; Deber, Charles M

    2017-04-01

    Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein-protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane-insertable peptides for the disruption or stabilization of membrane-based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non-natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane-insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane-based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  13. [Effect of electron irradiation on the structure of microsomal membrane proteins].

    PubMed

    Gorbenko, G P; Diubko, T S; Krupin, V D

    1998-01-01

    Using tryptophan fluorescence quenching by iodide and acrylamide the effect of electrons with the energy 5 Mev on the structure of microsomal membrane proteins has been studied. Conformation of microsomal proteins were found to change upon irradiation.

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

    SciTech Connect

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

    1992-01-14

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

  15. Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles.

    PubMed

    Moon, Dong Chan; Choi, Chul Hee; Lee, Jung Hwa; Choi, Chi-Won; Kim, Hye-Yeon; Park, Jeong Soon; Kim, Seung Il; Lee, Je Chul

    2012-02-01

    Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

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

    PubMed

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

    2012-08-01

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

  17. An Integrated Framework Advancing Membrane Protein Modeling and Design

    PubMed Central

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

    2015-01-01

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

  18. Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

    PubMed Central

    Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

    1998-01-01

    The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

  19. Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy.

    PubMed

    Itaya, Miki; Brett, Ian C; Smith, Steven O

    2012-01-01

    Membrane proteins function as receptors, channels, transporters, and enzymes. These proteins are generally difficult to express and purify in a functional form due to the hydrophobic nature of their membrane spanning sequences. Studies on membrane proteins with a single membrane spanning helix have been particularly challenging. Single-pass membrane proteins will often form dimers or higher order oligomers in cell membranes as a result of sequence motifs that mediate specific transmembrane helix interactions. Understanding the structural basis for helix association provides insights into how these proteins function. Nevertheless, nonspecific association or aggregation of hydrophobic membrane spanning sequences can occur when isolated transmembrane domains are reconstituted into membrane bilayers or solubilized into detergent micelles for structural studies by solid-state or solution NMR spectroscopy. Here, we outline the methods used to synthesize, purify, and characterize single transmembrane segments for structural studies. Two synthetic strategies are discussed. The first strategy is to express hydrophobic peptides as protein chimera attached to the maltose binding protein. The second strategy is by direct chemical synthesis. Purification is carried out by several complementary chromatography methods. The peptides are solubilized in detergent for solution NMR studies or reconstituted into model membranes for solid-state NMR studies. We describe the methods used to characterize the reconstitution of these systems prior to NMR structural studies to establish if there is nonspecific aggregation.

  20. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    NASA Astrophysics Data System (ADS)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

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

    PubMed Central

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

    2006-01-01

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

  2. Detergent-Specific Membrane Protein Crystallization Screens

    NASA Technical Reports Server (NTRS)

    Wiener, Michael

    2007-01-01

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

  3. Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

    PubMed

    Piry, A; Heino, A; Kühnl, W; Grein, T; Ripperger, S; Kulozik, U

    2012-04-01

    We investigated the fractionation of casein micelles and the whey protein β-lactoglobulin (β-LG) of skim milk by crossflow microfiltration (0.1 μm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and β-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for β-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

  4. Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during Sendai virus membrane fusion. II. Kinetics of bacteriophage. lambda. DNA injection

    SciTech Connect

    Novick, S.L.

    1990-01-01

    Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator 3-(trifluoromethyl)-3-(m({sup 125}I) iodophenyl)diazirine. During Sendai virus fusion with liposomes composed of cardiolipin or phosphatidylserine, the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F{sub 1} subunit with the target membrane is an initiating event in fusion. Correlation of hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. The F{sub 1} subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and the F{sub 2} subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions. Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion.

  5. The Use of Detergents to Purify Membrane Proteins.

    PubMed

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

    2016-04-01

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

  6. Valosin-containing Protein-interacting Membrane Protein (VIMP) Links the Endoplasmic Reticulum with Microtubules in Concert with Cytoskeleton-linking Membrane Protein (CLIMP)-63*

    PubMed Central

    Noda, Chikano; Kimura, Hana; Arasaki, Kohei; Matsushita, Mitsuru; Yamamoto, Akitsugu; Wakana, Yuichi; Inoue, Hiroki; Tagaya, Mitsuo

    2014-01-01

    The distribution and morphology of the endoplasmic reticulum (ER) in mammalian cells depend on both dynamic and static interactions of ER membrane proteins with microtubules (MTs). Cytoskeleton-linking membrane protein (CLIMP)-63 is exclusively localized in sheet-like ER membranes, typical structures of the rough ER, and plays a pivotal role in the static interaction with MTs. Our previous study showed that the 42-kDa ER-residing form of syntaxin 5 (Syn5L) regulates ER structure through the interactions with both CLIMP-63 and MTs. Here, we extend our previous study and show that the valosin-containing protein/p97-interacting membrane protein (VIMP)/SelS is also a member of the family of proteins that shape the ER by interacting with MTs. Depletion of VIMP causes the spreading of the ER to the cell periphery and affects an MT-dependent process on the ER. Although VIMP can interact with CLIMP-63 and Syn5L, it does not interact with MT-binding ER proteins (such as Reep1) that shape the tubular smooth ER, suggesting that different sets of MT-binding ER proteins are used to organize different ER subdomains. PMID:25008318

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

    PubMed Central

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

    2000-01-01

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

  8. Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles.

    PubMed

    Wiser, M F; Sartorelli, A C; Patton, C L

    1990-01-01

    Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.

  9. Higher-order assemblies of BAR domain proteins for shaping membranes.

    PubMed

    Suetsugu, Shiro

    2016-06-01

    Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed.

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

    PubMed

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

    2016-04-01

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

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

    PubMed

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

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

  12. Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

    PubMed

    Coïc, Yves-Marie; Vincent, Michel; Gallay, Jacques; Baleux, Françoise; Mousson, Florence; Beswick, Veronica; Neumann, Jean-Michel; de Foresta, Béatrice

    2005-12-01

    Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We

  13. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    NASA Astrophysics Data System (ADS)

    Kondrashkina, E.; Khvostichenko, D. S.; Perry, S. L.; Von Osinski, J.; Kenis, P. J. A.; Brister, K.

    2013-03-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A-1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.

  14. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    SciTech Connect

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  15. Membrane Interacting Regions of Dengue Virus NS2A Protein

    PubMed Central

    2015-01-01

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

  16. Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: a study with bio-orthogonal chemical probes.

    PubMed

    Ciana, Annarita; Achilli, Cesare; Hannoush, Rami N; Risso, Angela; Balduini, Cesare; Minetti, Giampaolo

    2013-03-01

    Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.

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

    PubMed

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

    1976-03-05

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

  18. The role of hydrophobic interactions in positioning of peripheral proteins in membranes

    PubMed Central

    Lomize, Andrei L; Pogozheva, Irina D; Lomize, Mikhail A; Mosberg, Henry I

    2007-01-01

    Background Three-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined. Results We report the first large-scale computational study of monotopic/peripheral proteins with known 3D structures. The optimal translational and rotational positions of 476 proteins are determined by minimizing energy of protein transfer from water to the lipid bilayer, which is approximated by a hydrocarbon slab with a decadiene-like polarity and interfacial regions characterized by water-permeation profiles. Predicted membrane-binding sites, protein tilt angles and membrane penetration depths are consistent with spin-labeling, chemical modification, fluorescence, NMR, mutagenesis, and other experimental studies of 53 peripheral proteins and peptides. Experimental membrane binding affinities of peripheral proteins were reproduced in cases that did not involve a helix-coil transition, specific binding of lipids, or a predominantly electrostatic association. Coordinates of all examined peripheral proteins and peptides with the calculated hydrophobic membrane boundaries, subcellular localization, topology, structural classification, and experimental references are available through the Orientations of Proteins in Membranes (OPM) database. Conclusion Positions of diverse peripheral proteins and peptides in the lipid bilayer can be accurately predicted using their 3D structures that represent a proper membrane-bound conformation and oligomeric state, and have membrane binding elements present. The success of the implicit solvation model suggests that hydrophobic interactions are usually sufficient to determine the spatial position of a protein in the membrane, even when electrostatic interactions or specific binding of lipids are substantial. Our results demonstrate that

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

    NASA Astrophysics Data System (ADS)

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

    2004-03-01

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

  20. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity.

    PubMed

    Watson, Eleanor; Sherry, Aileen; Inglis, Neil F; Lainson, Alex; Jyothi, Dushyanth; Yaga, Raja; Manson, Erin; Imrie, Lisa; Everest, Paul; Smith, David G E

    2014-09-01

    Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  1. Size-dependent protein segregation at membrane interfaces

    NASA Astrophysics Data System (ADS)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  2. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  3. Directly probing rapid membrane protein dynamics with an atomic force microscope: a study of light-induced conformational alterations in bacteriorhodopsin.

    PubMed

    Lewis, A; Rousso, I; Khachatryan, E; Brodsky, I; Lieberman, K; Sheves, M

    1996-05-01

    This paper demonstrates that an atomic force microscope can be used to directly monitor rapid membrane protein dynamics. For this demonstration the membrane-bound proton pump, bacteriorhodopsin, has been investigated. It has been unequivocally shown that the light-induced dynamic alterations that have been observed do not arise from external artifacts such as heating of the sample by the incident light, but that these changes can be directly linked to the light-induced protein conformational alterations in this membrane. In essence, it has been shown that the light energy absorbed by bacteriorhodopsin is converted not only to chemical energy but also to mechanical energy. In summary a new ultrasensitive tool is described for monitoring the molecular dynamics of materials with wide applicability to fundamental and applied science.

  4. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4% SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.

  5. Assembly of outer-membrane proteins in bacteria and mitochondria.

    PubMed

    Tommassen, Jan

    2010-09-01

    The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic alpha-helices, integral outer-membrane proteins (OMPs) form beta-barrels. Similar beta-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these beta-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of beta-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial beta-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

  6. Solubilization of a membrane protein by combinatorial supercharging.

    PubMed

    Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

    2011-04-15

    Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

  7. Biomolecular Stress-Sensitive Gauges: Surface-Mediated Immobilization of Mechanosensitive Membrane Protein

    DTIC Science & Technology

    2003-01-01

    Immobilization of Mechanosensitive Membrane Protein 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER...8-98) Prescribed by ANSI Std Z39-18 Biomolecular Stress-Sensitive Gauges: Surface-Mediated Immobilization of Mechanosensitive Membrane Protein...biomolecular gauges.1 Studies of a mechanosensitive protein of large conductance (MscL) had shown that a dramatic change in the protein conformation

  8. Basement membrane proteins in the space of Disse: a reappraisal.

    PubMed Central

    Griffiths, M R; Keir, S; Burt, A D

    1991-01-01

    The distribution of two major basement membrane components, type IV collagen and laminin, was studied within the perisinusoidal space of Disse in normal human liver using (i) an immunoperoxidase method for light microscopy and (ii) immunogold labelling for ultrastructural localisation. Although immunoreactivity depended on the mode of tissue fixation, both proteins could be identified at this site using a panel of affinity purified antibodies. These findings indicate that these proteins are normal constituents of the perisinusoidal extracellular matrix, and refute the hypothesis that capillarization of the sinusoids in chronic liver disease results from neo-expression of laminin in the space of Disse. Images PMID:1890197

  9. Effect of membrane protein concentration on binding of /sup 3/H-imipramine in human platelets

    SciTech Connect

    Barkai, A.I.; Kowalik, S.; Baron, M.

    1985-02-01

    Binding of /sup 3/H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing /sup 3/H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The /sup 3/H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies.

  10. Relationship between a stress membrane protein of Oenococcus oeni and glyceraldehyde-3-phosphate dehydrogenases.

    PubMed

    Carreté, Ramon; Reguant, Cristina; Bordons, Albert; Constantí, Magda

    2005-10-01

    The goal of this study was to analyze how the profiles of membrane proteins of Oenococcus oeni change under particular stress conditions of wine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis protein profiles of membrane fraction showed that a 40-kDa protein was overexpressed in the presence of SO2. The sequence of its N-terminal fragment showed a significant identity with glyceraldehyde-3-phosphate dehydrogenases (GAPDHs), but the protein showed no GAPDH activity. This sequence was compared with those of other GAPDHs with ClustalW alignment, and it was found to be somewhat similar to that of the cell-wall and membrane proteins of other lactic acid bacteria.

  11. Disturbed vesicular trafficking of membrane proteins in prion disease.

    PubMed

    Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

    Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

  13. Crystallizing Membrane Proteins in Lipidic Mesophases. A Host Lipid Screen

    SciTech Connect

    Li, Dianfan; Lee, Jean; Caffrey, Martin

    2011-11-30

    The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. There is no good reason, however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. Thus, it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile. For this, monoacylglycerols with differing acyl chain characteristics, such as length and olefinic bond position, must be available. A lipid synthesis and purification program is in place in the author's laboratory to serve this need. In the current study with the outer membrane sugar transporter, OprB, we demonstrate the utility of host lipid screening as a means for generating diffraction-quality crystals. Host lipid screening is likely to prove a generally useful strategy for mesophase-based crystallization of membrane proteins.

  14. Sorting of streptavidin protein coats on phase-separating model membranes.

    PubMed

    Manley, Suliana; Horton, Margaret R; Lecszynski, Szymon; Gast, Alice P

    2008-09-01

    Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.

  15. Rotation of plasma membrane proteins measured by polarized fluorescence depletion

    NASA Astrophysics Data System (ADS)

    Barisas, B. George; Rahman, Noorul A.; Yoshida, Thomas M.; Roess, Deborah A.

    1990-05-01

    We have implemented a new laser microscopic method, polarized fluorescence depletion (PFD), for measuring the rotational dynamics of functional membrane proteins on individual, microscopically selected cells under physiological conditions. This method combines the long lifetimes of triplet-state probes with the sensitivity of fluorescence detection to measure macromolecular rotational correlation times from 10 microsec to > 1 ms. As examples, the rotational correlation time of Fc receptors (FcR) on the surface of 2H3 rat basophilic leukemia cells is 79.9 4.4 microsec at 4°C when labeled with eosin conjugates of IgE. This value is consistent with the known 100 kDa receptor size. When labeled with intact F4 anti-FcR monoclonal antibody, the rotational correlation time for FcER is increased about 2-fold to 170.8 +/- 6.5 microsec, consistent with receptor dimer formation on the plasma membrane and with the ability of this antibody to form FcER dimers on 2H3 cell surfaces. We have also examined the rotational diffusion of the luteinizing hormone receptor on plasma membranes of small ovine luteal cells. Luteinizing hormone receptors (LHR), when occupied by ovine luteinizing hormone (oLH), have a rotational correlation time of 20.5 +/- 0.1 microsec at 4°C. When occupied by human chorionic gonadotropin (hCG), LHR have a rotational correlation time of 46.2 +/- 0.4 microsec suggesting that binding of hCG triggers additional LHR interactions with plasma membrane proteins. Together these studies suggest the utility of PFD measurements in assessing molecular size and molecular association of membrane proteins on individual cells. Relative advantages of time- and frequency-domain implementations of PFD are also discussed.

  16. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

    PubMed

    Cahill, Bethaney K; Seeley, Kent W; Gutel, Dedra; Ellis, Terri N

    2015-11-01

    Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

  17. Probing Single Membrane Proteins by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Scheuring, S.; Sapra, K. Tanuj; Müller, Daniel J.

    In this book chapter, we describe the working principle of the atomic force microscope (AFM), followed by the applications of AFM in high-resolution imaging and single-molecule force spectroscopy of membrane proteins. In the imaging mode, AFM allows observing the assembly of membrane proteins directly in native membranes approaching a resolution of ~0.5 nm with an outstanding signal-to-noise ratio. Conformational deviations of individual membrane proteins can be observed and their functional states directly imaged. Time-lapse AFM can image membrane proteins at work. In conjunction with high- resolution imaging, the use of the AFM as a single-molecule force spectroscope (SMFS) has gained tremendous importance in recent years. This combination allows to locate the inter- and intramolecular interactions of single membrane proteins. SMFS allows characterization of interactions that guide the folding of proteins and describe the parameters that lead to their destabilization, malfunction and misfolding. Moreover, it enables to measure the interactions established by ligand- and inhibitor-binding and in membrane protein assemblies. Because of its practical use in characterizing various parameters of membrane proteins in their native environment, AFM can be aptly described as a `lab on a tip' device.

  18. Membrane Composition Variation and Underdamped Mechanics near Transmembrane Proteins and Coats

    NASA Astrophysics Data System (ADS)

    Rautu, S. Alex; Rowlands, George; Turner, Matthew S.

    2015-03-01

    We study the effect of transmembrane proteins on the shape, composition, and thermodynamic stability of the surrounding membrane. When the coupling between membrane composition and curvature is strong enough, the nearby membrane composition and shape both undergo a transition from overdamped to underdamped spatial variation, well before the membrane becomes unstable in the bulk. This transition is associated with a change in the sign of the thermodynamic energy and, hence, favors the early stages of coat assembly necessary for vesiculation (budding) and may suppress the activity of mechanosensitive membrane channels and transporters. Our results suggest an approach to obtain physical parameters of the membrane that are otherwise difficult to measure.

  19. Study on the resistance mechanism via outer membrane protein OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa.

    PubMed

    Cai, Shuangqi; Chen, Yiqiang; Song, Dezhi; Kong, Jinliang; Wu, Yanbin; Lu, Huasong

    2016-11-01

    The aim of the present study was to evaluate the imipenem-resistant mechanism via the outer membrane protein (OMP) OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The Pseudomonas aeruginosa was clinically separated and validated by VITEK-2 full-automatic bacteria analyzer. Drug resistance, sensitive antibiotics and minimum inhibitory concentration (MIC) were tested using the drug sensitivity analysis system. The phenotype positive strains of MBL genes were screened using the Kirby-Bauer diffusion method by adding metal ion-chelating agent EDTA on the imipenem susceptibility paper. IMP-1, VIM-1 and SPM metaloenzyme genes were tested by polymerase chain reaction (PCR)-telomeric repeat amplification protocol (TRAP). The OMP OprD2 genes were tested by PCR-TRAP, and the protein expression was tested using western blot analysis. The location of OMP OprD2 was confirmed using the sodium salicylate inhibition test. The results showed that 80 portions (40%) of MBL-positive strains were screened out of 200 specimens. Imipenem-resistant Pseudomonas aeruginosa (IRPA) and MIC values were significantly higher than quality control bacteria and control bacteria (P<0.05). A total of 35 cases with IMP-1 positive, 20 with VIM-1 positive, 16 with SPM positive, 5 with 2 positive genes and 4 with 3 positive genes were screened among MBL positive strains. A total of 150 portions (75%) of OprD2 deficiencies were screened from 200 specimens. The standard strains and sensitive strains showed OprD2 protein bands at 45 kDa while no OprD2 protein bands appeared in OprD2 deficiency strains. It was in accordance with gene detection. In conclusion, OMP OprD2 deficiency and MBL phenotype positivity may be important mechanisms of IRPA.

  20. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

    PubMed

    Rood, Ilse M; Merchant, Michael L; Wilkey, Daniel W; Zhang, Terry; Zabrouskov, Vlad; van der Vlag, Johan; Dijkman, Henry B; Willemsen, Brigith K; Wetzels, Jack F; Klein, Jon B; Deegens, Jeroen K

    2015-11-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

  1. Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids.

    PubMed

    Baumgart, Tobias; Capraro, Benjamin R; Zhu, Chen; Das, Sovan L

    2011-01-01

    Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations.

  2. Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

    SciTech Connect

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis; Becker, Jeffrey Marvin; Heller, William T

    2008-01-01

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  3. Fluorescence polarization studies of rat intestinal microvillus membranes.

    PubMed Central

    Schachter, D; Shinitzky, M

    1977-01-01

    Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin. PMID:14174

  4. Protein quality control at the inner nuclear membrane.

    PubMed

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

    2014-12-18

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

  5. Characterization of membrane protein interactions by isothermal titration calorimetry.

    PubMed

    Situ, Alan J; Schmidt, Thomas; Mazumder, Parichita; Ulmer, Tobias S

    2014-10-23

    Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix-helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of -4.61±0.04kcal/mol at bicelle conditions where the sampling of random helix-helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of >1kcal/mol was obtained from helix-helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500±100s(-1) and 2.1±0.1s(-1), respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.

  6. Characterization of protein-membrane binding interactions via a microplate assay employing whole liposome immobilization.

    PubMed

    Smith, Matthew D; Best, Michael D

    2011-01-01

    Protein-cell membrane binding interactions control numerous vital biological processes, many of which can go awry during disease onset. However, the study of these events is complicated by the complexity of the membrane bilayer. These efforts would benefit from a rapid and easily accessible method for characterizing protein-membrane recognition events. Herein, we describe a microplate-based method for the detection of protein-membrane binding that employs whole liposome immobilization using a biotin anchor. First, control studies are detailed to test for nonspecific liposome immobilization (fluorescence assay; see Subheading 3.2), and to ensure that liposomes remain intact on the microplate surface (dye leakage assay; see Subheading 3.3). Finally, a protein-membrane binding detection assay is described through the example of protein kinase Cα binding to surface-immobilized whole liposomes (see Subheading 3.4).

  7. Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT.

    PubMed

    Belzile, Olivier; Hernandez-Lara, Carmen I; Wang, Qian; Snell, William J

    2013-08-05

    The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.

  8. BPROMPT: A consensus server for membrane protein prediction.

    PubMed

    Taylor, Paul D; Attwood, Teresa K; Flower, Darren R

    2003-07-01

    Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT.

  9. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

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

    PubMed Central

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

    2013-01-01

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

  11. bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

    PubMed Central

    Zheng, Hui; Lee, Sungsoo; Llaguno, Marc C.

    2016-01-01

    Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted

  12. Formation of assemblies on cell membranes by secreted proteins: molecular studies of free λ light chain aggregates found on the surface of myeloma cells.

    PubMed

    Hutchinson, Andrew T; Malik, Ansha; Berkahn, Mark B; Agostino, Mark; To, Joyce; Tacchi, Jessica L; Djordjevic, Steven P; Turnbull, Lynne; Whitchurch, Cynthia B; Edmundson, Allen B; Jones, Darren R; Raison, Robert L; Ramsland, Paul A

    2013-09-15

    We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

  13. New vehicles allow detergent-free mass spectrometry of membrane protein complexes

    PubMed Central

    Hopper, Jonathan T.S.; Yu, Yvonne Ting-Chun; Li, Dianfan; Raymond, Alison; Bostock, Mark; Liko, Idlir; Mikhailov, Victor; Laganowsky, Arthur; Benesch, Justin L.P.; Caffrey, Martin; Nietlispach, Daniel; Robinson, Carol V.

    2013-01-01

    The rapid growth in the study of membrane protein complexes in lipid microenvironments prompts new methods for their characterization. Here we develop mass spectrometry as a method for studying membrane proteins incorporated into amphipols, bicelles and nanodiscs. We compare these detergent-free vehicles with micelles for their ability to preserve the interactions of oligomeric complexes and to stabilize proteins that require defined lipid environments. PMID:24122040

  14. Membrane protein production in Escherichia coli cell-free lysates.

    PubMed

    Henrich, Erik; Hein, Christopher; Dötsch, Volker; Bernhard, Frank

    2015-07-08

    Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

  15. A gas chromatography-mass spectrometry method to monitor detergents removal from a membrane protein sample.

    PubMed

    Shi, Chaowei; Han, Fang; Xiong, Ying; Tian, Changlin

    2009-12-01

    In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography-mass spectrometry selected ion monitoring (GC-MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC-MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.

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

    PubMed

    Ashe, Mark P; Bill, Roslyn M

    2011-06-01

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

  17. A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.

    PubMed

    Barret, Laurie-Anne; Polidori, Ange; Bonneté, Françoise; Bernard-Savary, Pierre; Jungas, Colette

    2013-03-15

    The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 μg of DDM in water with a limit of detection of 0.05 μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements.

  18. Isolation of a unique membrane protein from Naegleria fowleri.

    PubMed

    Réveiller, F L; Suh, S J; Sullivan, K; Cabanes, P A; Marciano-Cabral, F

    2001-01-01

    Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.

  19. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  20. Membrane protein insertion: mixing eukaryotic and prokaryotic concepts.

    PubMed

    Schleiff, Enrico; Soll, Jürgen

    2005-11-01

    Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.

  1. Metaproteomic analysis of biocake proteins to understand membrane fouling in a submerged membrane bioreactor.

    PubMed

    Zhou, Zhongbo; Meng, Fangang; He, Xiang; Chae, So-Ryong; An, Yujia; Jia, Xiaoshan

    2015-01-20

    Metaproteomic analyses, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)/TOF mass spectrometer (MS) detection, were used to trace and identify biocake proteins on membranes in a bench-scale submerged membrane bioreactor (MBR). 2D-PAGE images showed that proteins in the biocake (S3) at a low transmembrane pressure (TMP) level (i.e., before the TMP jump) had larger gray intensities in the pH 5.5–7.0 region regardless of the membrane flux, similar to soluble microbial product (SMP) proteins. However, the biocake (S2 and S4) at a high TMP level (i.e., after the TMP jump) had many more proteins in the pH range of 4.0–5.5, similar to extracellular polymeric substance (EPS) proteins. Such similarities between biocake proteins and SMP or EPS proteins can be useful for tracing the sources of proteins resulting in membrane fouling. In total, 183 differentially abundant protein spots were marked in the three biocakes (S2, S3, and S4). However, only 32 protein spots co-occurred in the 2D gels of the three biocakes, indicating that membrane fluxes and TMP evolution levels had significant effects on the abundance of biocake proteins. On the basis of the MS and MS/MS data, 23 of 71 protein spots were successfully identified. Of the 23 proteins, outer membrane proteins (Omp) were a major contributor (60.87%). These Omps were mainly from potential surface colonizers such as Aeromonas, Enterobacter, Pseudomonas, and Thauera. Generally, the metaproteomic analysis is a useful alternative to trace the sources and compositions of biocake proteins on the levels of molecules and bacteria species that can provide new insight into membrane fouling.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-Bin; Ye, Jin-Zhou; Yang, Man-Jun; Jiang, Ming; Peng, Xuan-Xian

    2017-01-01

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

  4. Practical aspects in expression and purification of membrane proteins for structural analysis.

    PubMed

    Vinothkumar, Kutti R; Edwards, Patricia C; Standfuss, Joerg

    2013-01-01

    A surge of membrane protein structures in the last few years can be attributed to advances in technologies starting at the level of genomes, to highly efficient expression systems, stabilizing conformational flexibility, automation of crystallization and data collection for screening large numbers of crystals and the microfocus beam lines at synchrotrons. The substantial medical importance of many membrane proteins provides a strong incentive to understand them at the molecular level. It is becoming obvious that the major bottleneck in many of the membrane projects is obtaining sufficient amount of stable functional proteins in a detergent micelle for structural studies. Naturally, large effort has been spent on optimizing and advancing multiple expression systems and purification strategies that have started to yield sufficient protein and structures. We describe in this chapter protocols to refold membrane proteins from inclusion bodies, purification from inner membranes of Escherichia coli and from mammalian cell lines.

  5. Host membrane proteins involved in the replication of tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Miyashita, Shuhei; Katoh, Etsuko; Ishikawa, Masayuki

    2012-12-01

    Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

  6. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

    PubMed Central

    Shen, Hsin-Hui; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

    2015-01-01

    In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here, we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines. PMID:25341963

  7. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

    NASA Astrophysics Data System (ADS)

    Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

    2014-10-01

    In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

  8. MALDI tissue profiling of integral membrane proteins from ocular tissues.

    PubMed

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

    2008-06-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this article, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed age related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods.

  9. Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona.

    PubMed

    Silva-Briano, Marcelo; Martínez-Hernández, Sandra Luz; Adabache-Ortíz, Araceli; Ventura-Juárez, Javier; Salinas, Eva; Quintanar, J Luis

    2007-08-01

    Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.

  10. The Membrane Permeability Outcome study.

    PubMed

    Locatelli, Francesco; Cavalli, Andrea; Manzoni, Celestina; Pontoriero, Giuseppe

    2011-01-01

    Many observational studies have consistently shown that high-flux hemodialysis has positive effects on the survival and morbidity of uremic patients when compared with low-flux hemodialysis. However, the HEMO study, a randomized trial designed to evaluate the effect of membrane permeability on patient survival, showed only an 8% non-statistically significant reduction of mortality, albeit a secondary analysis suggested an advantage for high-flux membranes in certain patient subgroups. The prospective, randomized Membrane Permeability Outcome (MPO) study investigated the impact of membrane permeability on survival in incident hemodialysis patients who had low albumin (≤4 g/dl) and normal albumin ( >4 g/dl) as separate randomization groups. Patients with serum albumin ≤4 g/dl had significantly better survival rates in the high-flux group compared with the low-flux group (p = 0.032). Moreover, a post-hoc secondary analysis showed that high-flux membranes may significantly improve survival in diabetic patients. No difference was found in patients with normal albumin levels. Considering the increasing number of dialysis patients with low serum albumin levels and with diabetes, the relevance of the MPO study led to the publication of a position statement by the European Renal Best Practice Advisory Board. This board strongly recommended that high-flux hemodialysis should be used for high-risk patients and, with a lower degree of evidence, even also for low-risk subjects due to the substantial reduction in β(2)-microglobulin levels observed in the high-flux group.

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

    PubMed Central

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

    2016-01-01

    We demonstrate that dynamic nuclear polarization (DNP) of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of 6-fold for the dimeric protein. The enhancement affect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  12. Proteomic characterization of human milk fat globule membrane proteins during a 12 month lactation period.

    PubMed

    Liao, Yalin; Alvarado, Rudy; Phinney, Brett; Lönnerdal, Bo

    2011-08-05

    The milk fat globule membrane (MFGM) contains proteins which have been implicated in a variety of health benefits. Milk fat globule membrane proteins were isolated from human milk during a 12 month lactation period and subjected to in-solution digestion and liquid chromatography tandem mass spectrometry analysis. Data were pooled, and our results showed that 191 proteins were identified. Relative quantification of the identified MFGM proteins during the course of lactation was performed by label free spectral counting and differentiation expression analysis, which showed some proteins decreasing during the course of lactation whereas some increased or remained at a relatively constant level. The human MFGM proteins are distributed between intracellular, extracellular, and membrane-associated proteins, and they are mainly involved in cell communication and signal transduction, immune function, metabolism and energy production. This study provides more insights into the dynamic composition of human MFGM proteins, which in turn will enhance our understanding of the physiological significance of MFGM proteins.

  13. Studies of the Mu-Opioid Receptor/G-protein Complex Affinity Co-Purified and Membrane Preparations from 7315c Cells

    DTIC Science & Technology

    1989-06-09

    population of mu-opioid receptors. Morphine, an opioid agonist, inhibits prolactin secretion, in part, by inhibiting adenylyl cyclase activity via...interaction with multiple G-proteins in the 7315c cell membrane is that mu-opioid agonists inhibit prolactin secretion from this cell via the generation of...secretion. The receptor mediated activation of either Gil or Gi2 and subsequent inhibition of adenylyl cyclase activity would inhibit prolactin secretion

  14. The electrical interplay between proteins and lipids in membranes.

    PubMed

    Richens, Joanna L; Lane, Jordan S; Bramble, Jonathan P; O'Shea, Paul

    2015-09-01

    All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure. The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid-protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid-protein interactions.

  15. Genetically Encoded Protein Sensors of Membrane Potential.

    PubMed

    Storace, Douglas; Rad, Masoud Sepehri; Han, Zhou; Jin, Lei; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J; Sung, Uhna

    2015-01-01

    Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. However these dyes suffer from the drawbacks of non-specificity of cell staining and low accessibility of the dye to some cell types. Further progress in imaging activity is expected from the development of genetically encoded fluorescent sensors of membrane potential. Cell type specificity of expression of these fluorescent protein (FP) voltage sensors can be obtained via several different mechanisms. One is cell type specificity of infection by individual virus subtypes. A second mechanism is specificity of promoter expression in individual cell types. A third, depends on the offspring of transgenic animals with cell type specific expression of cre recombinase mated with an animal that has the DNA for the FP voltage sensor in all of its cells but its expression is dependent on the recombinase activity. Challenges remain. First, the response time constants of many of the new FP voltage sensors are slower (2-10 ms) than those of organic dyes. This results in a relatively small fractional fluorescence change, ΔF/F, for action potentials. Second, the largest signal presently available is only ~40% for a 100 mV depolarization and many of the new probes have signals that are substantially smaller. Large signals are especially important when attempting to detect fast events because the shorter measurement interval results in a relatively small number of detected photons and therefore a relatively large shot noise (see Chap. 1). Another kind of challenge has occurred when attempts were made to transition from one species to another or from one cell type to another or from cell culture to in vivo measurements.Several laboratories have recently described a number of novel FP voltage sensors. Here we attempt to critically review the current status of these developments in terms of signal size, time course, and in vivo function.

  16. Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

    PubMed Central

    Wang, Feng; Wu, Jiazhen; Gao, Jing; Liu, Shuheng; Jiang, Junguang; Jiang, Shibo; Wang, Hongda

    2014-01-01

    The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms. PMID:24806512

  17. The driving forces of membrane remodeling by non-intrinsically curved proteins

    NASA Astrophysics Data System (ADS)

    Ryan, Christopher J.; Stachowiak, Jeanne C.; Schmid, Eva M.; Fletcher, Daniel A.; Geissler, Phillip L.

    2011-03-01

    Membranes are dynamically remodeled during numerous processes essential to cells. Among the most well-studied effectors of this remodeling are BAR family proteins, which are small and have a banana-like intrinsic curvature that senses, forms, and stabilizes curved membranes without expending energy as ATP or GTP. Recent experiments in reduced systems have shown, however, that small proteins that feature no such intrinsic curvature can similarly cluster at and dramatically remodel membranes. These proteins have no distinguishing features other than their size and their membrane-binding sites, and the dominant effect that is driving curvature is not well understood. Here, we present a coarse-grained simulation study that captures protein steric and binding effects as well as membrane fluctuations at large scales. We use this model to systematically test for the role that such attributes play in the resulting dynamics and equilibrium structures of remodeling processes that feature this motif.

  18. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  19. Supported lipid bilayers as models for studying membrane domains.

    PubMed

    Kiessling, Volker; Yang, Sung-Tae; Tamm, Lukas K

    2015-01-01

    Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network.

  20. Probing Peptide and Protein Insertion in a Biomimetic S-Layer Supported Lipid Membrane Platform

    PubMed Central

    Damiati, Samar; Schrems, Angelika; Sinner, Eva-Kathrin; Sleytr, Uwe B.; Schuster, Bernhard

    2015-01-01

    The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided. PMID:25633104

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

    PubMed

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

    2012-09-01

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

  2. Single-Molecule Microscopy and Force Spectroscopy of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Engel, Andreas; Janovjak, Harald; Fotiadis, Dimtrios; Kedrov, Alexej; Cisneros, David; Müller, Daniel J.

    Single-molecule atomic force microscopy (AFM) provides novel ways to characterize the structure-function relationship of native membrane proteins. High-resolution AFM topographs allow observing the structure of single proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. We will review these feasibilities illustrating examples of membrane proteins in native and reconstituted membranes. Classification of individual topographs of single proteins allows understanding the principles of motions of their extrinsic domains, to learn about their local structural flexibilities and to find the entropy minima of certain conformations. Combined with the visualization of functionally related conformational changes these insights allow understanding why certain flexibilities are required for the protein to function and how structurally flexible regions allow certain conformational changes. Complementary to AFM imaging, single-molecule force spectroscopy (SMFS) experiments detect molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to measure interactions that stabilize secondary structures such as transmembrane α-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the locations of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes.

  3. Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry.

    PubMed

    Wang, Zhuowei; Wang, Min; Tong, Wei

    2010-12-01

    In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.

  4. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

    PubMed Central

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-01-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C10, was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrié et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca2+-dependent ATPase and Ca2+-pumping activity. We found that 18C10-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions. PMID:15459343

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Lu, Yang; Fan, Meng

    2017-01-01

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

  7. Dynamics of Membrane Proteins within Synthetic Polymer Membranes with Large Hydrophobic Mismatch.

    PubMed

    Itel, Fabian; Najer, Adrian; Palivan, Cornelia G; Meier, Wolfgang

    2015-06-10

    The functioning of biological membrane proteins (MPs) within synthetic block copolymer membranes is an intriguing phenomenon that is believed to offer great potential for applications in life and medical sciences and engineering. The question why biological MPs are able to function in this completely artificial environment is still unresolved by any experimental data. Here, we have analyzed the lateral diffusion properties of different sized MPs within poly(dimethylsiloxane) (PDMS)-containing amphiphilic block copolymer membranes of membrane thicknesses between 9 and 13 nm, which results in a hydrophobic mismatch between the membrane thickness and the size of the proteins of 3.3-7.1 nm (3.5-5 times). We show that the high flexibility of PDMS, which provides membrane fluidities similar to phospholipid bilayers, is the key-factor for MP incorporation.

  8. Protein binding properties of surface-modified porous polyethylene membranes.

    PubMed

    Greene, George; Radhakrishna, Harish; Tannenbaum, Rina

    2005-10-01

    In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI-PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI-PAA bilayer-modified PPE surfaces.

  9. Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes.

    PubMed

    Cheng, Xi; Jo, Sunhwan; Qi, Yifei; Marassi, Francesca M; Im, Wonpil

    2015-04-21

    Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys(40), residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein

  10. Dynamics and heterogeneity of bovine hippocampal membranes: role of cholesterol and proteins.

    PubMed

    Mukherjee, Soumi; Kombrabail, Mamata; Krishnamoorthy, G; Chattopadhyay, Amitabha

    2007-09-01

    The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.

  11. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  12. Overexpression of membrane proteins from higher eukaryotes in yeasts.

    PubMed

    Emmerstorfer, Anita; Wriessnegger, Tamara; Hirz, Melanie; Pichler, Harald

    2014-09-01

    Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.

  13. Human erythrocyte membrane proteins of zone 4.5 exist as families of related proteins.

    PubMed

    Whitfield, C F; Coleman, D B; Kay, M M; Shiffer, K A; Miller, J; Goodman, S R

    1985-01-01

    An analysis of the polypeptide composition of zone 4.5 of human erythrocyte membranes has been done by immunoautoradiographic and two-dimensional peptide mapping techniques. Results of these studies demonstrated that the Coomassie blue profile was constant, with 14 well-resolved bands present. Zone 4.5 polypeptides existed as at least four families of two or more components with closely related polypeptide backbones. The families could be distinguished on the basis of their extraction characteristics, immunological cross-reactivity, and two-dimensional peptide maps. One family was related to protein 4.1, one family was related to band 3, and two families were independent and not similar to other larger membrane proteins. The data show that all of the visualized bands in zone 4.5 do not have the same protein composition and that several closely related forms of some polypeptides are present.

  14. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  15. Rational design of a fusion partner for membrane protein expression in E. coli

    PubMed Central

    Luo, Jianying; Choulet, Julie; Samuelson, James C

    2009-01-01

    We have designed a novel protein fusion partner (P8CBD) to utilize the co-translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP-dependence was demonstrated by analyzing the membrane translocation of P8CBD-PhoA fusion proteins in wt and SRP-ffh77 mutant cells. We also demonstrate that the P8CBD N-terminal fusion partner promotes over-expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over-expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane-associated protein. PMID:19530231

  16. Protective effect of black tea on integral membrane proteins in rat liver.

    PubMed

    Szachowicz-Petelska, Barbara; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew

    2013-01-01

    Ethanol intoxication is accompanied by oxidative stress formation. Consequently, it leads to disturbances in cellular metabolism that can alter the structure and function of cell membrane components. Black tea displays antioxidant properties, protects membrane phospholipids and may protect integral membrane proteins. In the present study, we examined whether black tea induces changes in the liver integral membrane proteins of 12-months old rats chronically intoxicated with ethanol. To estimate qualitatively and quantitatively the levels of the liver integral membrane proteins, the proteins were selectively hydrolyzed by trypsin, the obtained peptides were resolved by HPLC and the levels of specific amino acids within the individual peptides were determined. All of the obtained peptides contained phenylalanine (Phe), cysteine (Cys) and lysine (Lys). Compared to the control group, rats in the ethanol intoxication group showed decreased liver levels of integral membrane proteins as well as fewer trypsin-hydrolyzed peptides and amino acids in the hydrolyzed peptides. Administration of black tea to ethanol-intoxicated rats partially protected proteins against the structural changes caused by ethanol. Black tea prevented decreases in the levels of cysteine (in about 90% of cases), lysine (in about 60% of cases), phenylalanine (in about 70% of cases) and examined peptides (in about 60% of cases). The liver protein level was higher (by about 18%) in rats who received black tea and ethanol than in those who received ethanol alone. In conclusion, black tea partially protects the composition and level of rat liver cell integral membrane proteins against changes caused by ethanol intoxication.

  17. Molecular interactions between proteins and synthetic membrane polymer films

    SciTech Connect

    Pincet, F.; Perez, E.; Belfort, G.

    1995-04-01

    To help understand the effects of protein adsorption on membrane filtration performance, we have measured the molecular interactions between cellulose acetate films and two proteins with different properties (ribonuclease A and human serum albumin) with a surface force apparatus. Comparison of forces between two protein layers with those between a protein layer and a cellulose acetate (CA) film shows that, at high pH, both proteins retained their native conformation on interacting with the CA film while at the isoelectric point (pI) or below the tertiary structure of proteins was disturbed. These measurements provide the first molecular evidence that disruption of protein tertiary structure could be responsible for the reduced permeation flows observed during membrane filtration of protein solutions and suggest that operating at high pH values away from the pI of proteins will reduce such fouling. 60 refs., 9 figs., 5 tabs.

  18. Prediction of buried helices in multispan alpha helical membrane proteins.

    PubMed

    Adamian, Larisa; Liang, Jie

    2006-04-01

    Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation. Given that the number of buried helices in a membrane protein is known, our method achieves an accuracy of 78% and a Matthew's correlation coefficient of 0.68. A server for this tool (RANTS) is available online at http://gila.bioengr.uic.edu/lab/.

  19. Curvature forces in membrane lipid-protein interactions.

    PubMed

    Brown, Michael F

    2012-12-11

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes in membrane proteins, involving folding, stability, and membrane shape transitions, potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function.

  20. Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.

    PubMed

    Petrosyan, Rafayel; Bippes, Christian A; Walheim, Stefan; Harder, Daniel; Fotiadis, Dimitrios; Schimmel, Thomas; Alsteens, David; Müller, Daniel J

    2015-05-13

    Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

  1. Repair of Nerve Cell Membrane Damage by Calcium-Dependent, Membrane-Binding Proteins (Revised)

    DTIC Science & Technology

    2012-09-01

    family exhibit a “ bivalent ” activity resulting in the aggregation of membranes coincident with the binding of the annexin to the membrane [7,8]. Such...10. [12] D.J. Selkoe, Alzheimer’s disease: genes , proteins, and therapy, Physiol Rev 81 (2001) 741-766. [13] A. Demuro, I. Parker, G.E. Stutzmann

  2. Heterogeneous interactome between Litopenaeus vannamei plasma proteins and Vibrio parahaemolyticus outer membrane proteins.

    PubMed

    Liu, Xiang; She, Xin-Tao; Zhu, Qing-Feng; Li, Hui; Peng, Xuan-Xian

    2013-01-01

    A great loss has been suffered by microbial infectious diseases under intensive shrimp farming in recent years. In this background, the understanding of shrimp innate immunity becomes an importantly scientific issue, but little is known about the heterogeneous protein-protein interaction between pathogenic cells and hosts, which is a key step for the invading microbes to infect internet organs through bloodstream. In the present study, bacterial outer membrane (OM) protein array and pull-down approaches are used to isolate both Vibrio parahaemolyticus OM proteins that bind to shrimp serum proteins and the shrimp serum proteins that interact with bacterial cells, respectively. Three interacting shrimp serum proteins, hemocyanin, β-1,3-glucan binding protein and LV_HP_RA36F08r and thirty interacting OM proteins were determined. They form 63 heterogeneous protein-protein interactions. Nine out of the 30 OM proteins were randomly demonstrated to be up-regulated or down-regulated when bacterial cells were cultured with shrimp sera, indicating the biological significance of the network. The interesting findings uncover the complexity of struggle between host immunity and bacterial infection. Compared with our previous report on heterogeneous interactome between fish grill and bacterial OM proteins, the present study further extends the investigation from lower vertebrates to invertebrates and develops a bacterial OM protein array to identify the OM proteins bound with shrimp serum proteins, which elevates the frequencies of the bound OM proteins. Our results highlight the way to determine and understand the heterogeneous interaction between hosts and microbes.

  3. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    PubMed Central

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  4. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

    PubMed

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-08-12

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

  5. Mutual diffusion of interacting membrane proteins.

    PubMed Central

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

    1989-01-01

    The generalized Stokes-Einstein equation is used, together with the two-dimensional pressure equation, to analyze mutual diffusion in concentrated membrane systems. These equations can be used to investigate the role that both direct and hydrodynamic interactions play in determining diffusive behavior. Here only direct interactions are explicitly incorporated into the theory at high densities; however, both direct and hydrodynamic interactions are analyzed for some dilute solutions. We look at diffusion in the presence of weak attractions, soft repulsions, and hard-core repulsions. It is found that, at low densities, attractions retard mutual diffusion while repulsions enhance it. Mechanistically, attractions tend to tether particles together and oppose the dissipation of gradients or fluctuations in concentration, while repulsions provide a driving force that pushes particles apart. At higher concentrations, changes in the structure of the fluid enhance mutual diffusion even in the presence of attractions. It is shown that the theoretical description of postelectrophoresis relaxation and fluorescence correlation spectroscopy experiments must be modified if interacting systems are studied. The effects of interactions on mutual diffusion coefficients have probably already been seen in postelectrophoresis relaxation experiments. PMID:2775829

  6. [Molecular interactions of membrane proteins and erythrocyte deformability].

    PubMed

    Boivin, P

    1984-06-01

    The structural and functional properties of the erythrocytic membrane constitute one of the essential elements of the red cell deformability. They intervene not only in the flexibility of the membrane, but also in the surface/volume relation and, through transmembrane exchanges, in the internal viscosity of the red cells. These properties depend essentially on the molecular composition of the elements which constitute the membrane, and on their interactions. The shape of the red cell and the flexibility of its membrane depend, to a great extent, on the membrane skeleton, whose main components are spectrin, actin, and protein 4.1. The spectrin basic molecule is a heterodimer, but there occur interactions between dimers in vitro as well as in vivo, which lead to the formation of tetrameric and oligomeric structures of higher complexity. Disturbances of these interactions, such as have been observed in pathological cases, lead to an instability of the membrane, a loss of membrane fragments, and a decrease in the surface/volume relation, with, as a consequence, a reduced deformability. The stability of the membrane skeleton also depends on the interactions between spectrin and protein 4.1. These interactions occur through a binding site on the beta chain of spectrin apparently close to actin and calmodulin binding sites. Other interactions occur between the hydrophobic segment of spectrin and membrane lipids. The cytoskeleton is bound to the transmembrane proteins: by ankyrin to the internal segment of protein band 3, and by protein 4.1 to a glycoprotein named glycoconnectin. There seems to exist other, more direct, lower affinity bindings between the cytoskeleton on the one hand, and band 3 and glycophorin transmembrane proteins on the other hand, whose lateral mobilities are modified when the structure of the skeleton is perturbed. The membrane proteins, which are in contact with the cytosol, interact with the cytosolic proteins, in particular with certain enzymes

  7. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

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

  8. Detergent selection for enhanced extraction of membrane proteins.

    PubMed

    Arachea, Buenafe T; Sun, Zhen; Potente, Nina; Malik, Radhika; Isailovic, Dragan; Viola, Ronald E

    2012-11-01

    Generating stable conditions for membrane proteins after extraction from their lipid bilayer environment is essential for subsequent characterization. Detergents are the most widely used means to obtain this stable environment; however, different types of membrane proteins have been found to require detergents with varying properties for optimal extraction efficiency and stability after extraction. The extraction profiles of several detergent types have been examined for membranes isolated from bacteria and yeast, and for a set of recombinant target proteins. The extraction efficiencies of these detergents increase at higher concentrations, and were shown to correlate with their respective CMC values. Two alkyl sugar detergents, octyl-β-d-glucoside (OG) and 5-cyclohexyl-1-pentyl-β-d-maltoside (Cymal-5), and a zwitterionic surfactant, N-decylphosphocholine (Fos-choline-10), were generally effective in the extraction of a broad range of membrane proteins. However, certain detergents were more effective than others in the extraction of specific classes of integral membrane proteins, offering guidelines for initial detergent selection. The differences in extraction efficiencies among this small set of detergents supports the value of detergent screening and optimization to increase the yields of targeted membrane proteins.

  9. Optimal separation of jojoba protein using membrane processes

    SciTech Connect

    Nabetani, Hiroshi; Abbott, T.P.; Kleiman, R.

    1995-05-01

    The efficiency of a pilot-scale membrane system for purifying and concentrating jojoba protein was estimated. In this system, a jojoba extract was first clarified with a microfiltration membrane. The clarified extract was diafiltrated and the protein was purified with an ultrafiltration membrane. Then the protein solution was concentrated with the ultrafiltration membrane. Permeate flux during microfiltration was essentially independent of solids concentration in the feed, in contrast with the permeate flux during ultrafiltration which was a function of protein concentration. Based on these results, a mathematical model which describes the batchwise concentration process with ultrafiltration membranes was developed. Using this model, the combination of batchwise concentration with diafiltration was optimized, and an industrial-scale process was designed. The effect of ethylenediaminetetraacetic acid (EDTA) on the performance of the membrane system was also investigated. The addition of EDTA increased the concentration of protein in the extract and improved the recovery of protein in the final products. The quality of the final product (color and solubility) was also improved. However, EDTA decreased permeate flux during ultrafiltration.

  10. Fundamental and Applied Studies of Polymer Membranes

    NASA Astrophysics Data System (ADS)

    Imbrogno, Joseph

    Four major areas have been studied in this research: 1) synthesizing novel monomers, e.g. chiral monomers, to produce new types of functionalized membranes for the biotechnology and pharmaceutical industries, 2) hydrophobic brush membranes for desalinating brackish water, sea water, and separating organics, 3) fundamental studies of water interactions at surfaces using sum frequency generation (SFG), and 4) discovering new surface chemistries that will control the growth and differentiation of stem cells. We have developed a novel synthesis method in order to increase the breadth of our high throughput screening library. This library was generated using maleimide chemistry to react a common methacrylate linker with a variety of different functions groups (R groups) in order to form new monomers that were grafted from the surface of PES ultrafiltration membranes. From this work, we discovered that the chirality of a membrane can affect performance when separating chiral feed streams. This effect was observed when filtering bovine serum albumin (BSA) and ovalbumin in a high salt phosphate buffered saline (PBS, 150 mM salt). The Phe grafted membranes showed a large difference in performance when filtering BSA with selectivity of 1.13 and 1.00 for (S) and (R) Phe, respectively. However, when filtering ovalbumin, the (S) and (R) modified surfaces showed selectivity of 2.06 and 2.31, respectively. The higher selectivity enantiomer switched for the two different proteins. Permeability when filtering BSA was 3.06 LMH kPa-1 and 4.31 LMH kPa -1 for (S)- and (R)- Phe, respectively, and 2.65 LMH kPa -1 and 2.10 LMH kPa-1 when filtering ovalbumin for (S)- and (R)- Phe, respectively. Additionally, these effects were no longer present when using a low salt phosphate buffer (PB, 10 mM salt). Since, to our knowledge, membrane chirality is not considered in current industrial systems, this discovery could have a large impact on the pharmaceutical and biotechnology industries. We

  11. Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

    PubMed

    Shirzad-Wasei, Nazhat; van Oostrum, Jenny; Bovee-Geurts, Petra H M; Kusters, Lisanne J A; Bosman, Giel J C G M; DeGrip, Willem J

    2015-08-01

    Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

  12. Determining the Topology of Membrane-Bound Proteins Using PEGylation.

    PubMed

    Howe, Vicky; Brown, Andrew J

    2017-01-01

    Biochemical methods can help elucidate the membrane topology of hydrophobic membrane proteins where X-ray crystallography is difficult or impractical, providing important structural data. Here, we describe the method of PEGylation, which uses a cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), to determine the cytosolic accessibility of introduced cysteine residues. This accessibility is visualized using Western blotting to detect a band shift that indicates cysteine labeling by mPEG. Using scanning cysteine mutagenesis, followed by PEGylation, one can map the accessibility of the introduced cysteines, hence inferring the membrane topology of the protein.We used PEGylation to determine the membrane topology of the sterol regulatory domain of a cholesterol synthesis enzyme, squalene monooxygenase, identifying that it is anchored to the membrane via a re-entrant loop.

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

    PubMed Central

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

    2015-01-01

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

  14. Curvature Forces in Membrane Lipid-Protein Interactions

    PubMed Central

    Brown, Michael F.

    2012-01-01

    Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes of membrane proteins—involving folding, stability, and membrane shape transitions—potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics, and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function. PMID:23163284

  15. The functions of tryptophan residues in membrane proteins

    SciTech Connect

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

    1994-08-01

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

  16. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  17. The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores.

    PubMed

    Alberto, François; Botella, Lucien; Carlin, Fréderic; Nguyen-The, Christophe; Broussolle, Véronique

    2005-12-15

    Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.

  18. Hydrodynamics of bilayer membranes with diffusing transmembrane proteins.

    PubMed

    Callan-Jones, Andrew; Durand, Marc; Fournier, Jean-Baptiste

    2016-02-14

    We consider the hydrodynamics of lipid bilayers containing transmembrane proteins of arbitrary shape. This biologically-motivated problem is relevant to the cell membrane, whose fluctuating dynamics play a key role in phenomena ranging from cell migration, intercellular transport, and cell communication. Using Onsager's variational principle, we derive the equations that govern the relaxation dynamics of the membrane shape, of the mass densities of the bilayer leaflets, and of the diffusing proteins' concentration. With our generic formalism, we obtain several results on membrane dynamics. We find that proteins that span the bilayer increase the intermonolayer friction coefficient. The renormalization, which can be significant, is in inverse proportion to the protein's mobility. Second, we find that asymmetric proteins couple to the membrane curvature and to the difference in monolayer densities. For practically all accessible membrane tensions (σ > 10(-8) N m(-1)) we show that the protein density is the slowest relaxing variable. Furthermore, its relaxation rate decreases at small wavelengths due to the coupling to curvature. We apply our formalism to the large-scale diffusion of a concentrated protein patch. We find that the diffusion profile is not self-similar, owing to the wavevector dependence of the effective diffusion coefficient.

  19. [Membrane fouling based on change of membrane characteristic parameters during ultrafiltration of protein].

    PubMed

    Wang, Xu-Dong; Zhang, Yin-Hui; Wang, Lei; Zhang, Hui-Hui; Xia, Si-Qing

    2014-11-01

    In order to further understand membrane fouling mechanism of various protein systems during ultrafiltration, polyethersulfone (PES) ultrafiltration membrane with relative molecular weight cut off of 50 x 10(3) was used, the ultrafiltration processes of three kinds of protein solution were investigated: lysozyme ( LYS), bovine serum albumin ( BSA), and LYS + BSA. Contact angle meter, field emission scanning electron microscope (FESEM) and atomic force microscope (AFM) were adopted to determine the change of membrane characteristic parameters at different fouling stages. The results indicated that the changes of ultrafiltration membrane flux obviously exhibited three stages: sharp flux decline in the initial stage (approximately between 0-5 min), slow flux decline during the transition stage (approximately between 5-60 min), and stable flux in the late stage (approximately between 60-120 min). During the whole ultrafiltration process, the LYS-fouled membrane had the largest flux decline, followed by the LYS + BSA-fouled membrane, and the BSA-fouled membrane had the least decline. The changes of membrane characteristic parameters clearly indicated that the initial filtration stage of LYS was controlled by pore constriction, while pore blocking and pore constriction were the main fouling mechanism at the transition stage. Pore blocking was the main fouling mechanism of BSA in the initial fouling stage, while the transition stage was controlled by pore constriction. Cake filtration was the main fouling mechanism of LYS and BSA in the late stage. The membrane fouling of binary mixtures LYS + BSA appeared to be dominated by LYS.

  20. Effect of natural polymorphism on structure and function of the Yersinia pestis outer membrane porin F (OmpF protein): a computational study.

    PubMed

    Shaban, Hiba; Na, Insing; Kislichkina, Angelina A; Dentovskaya, Svetlana V; Anisimov, Andrey P; Uversky, Vladimir N

    2016-09-03

    The Yersinia pestis outer membrane porin F (OmpF) is a transmembrane protein located in the outer membrane of this Gram-negative bacterium which is the causative agent of plague, where it plays a significant role in controlling the selective permeability of the membrane. The amino acid sequences of OmpF proteins from 48 Y. pestis strains representing all currently available phylogenetic groups of this Gram-negative bacterium were recently deduced. Comparison of these amino acid sequences revealed that the OmpF can be present in four isoforms, the pestis-pestis type, and the pestis-microtus types I, II, and III. OmpF of the most recent pestis-pestis type has an alanine residue at the position 148, where all the pestis-microtus types have threonine there (T148A polymorphism). The variability of different pestis-microtus types is caused by an additional polymorphism at the 193rd position, where the OmpFs of the pestis-microtus type II and type III have isoleucine-glycine (IG(+)193) or isoleucine-glycine-isoleucine-glycine (IGIG(+)193) insertions, respectively (IG(+)193 and IGIG(+)193 polymorphism). To investigate potential effects of these sequence polymorphisms on the structural properties of the OmpF protein, we conducted multi-level computational analysis of its isoforms. Analysis of the I-TASSER-generated 3D-models revealed that the Yersinia OmpF is very similar to other non-specific enterobacterial porins. The T148A polymorphism affected a loop located in the external vestibule of the OmpF channel, whereas IG(+)193 and IGIG(+)193 polymorphisms affected one of its β-strands. Our analysis also suggested that polymorphism has moderate effect on the predicted local intrinsic disorder predisposition of OmpF, but might have some functional implementations.

  1. Lipids and topological rules governing membrane protein assembly☆

    PubMed Central

    Bogdanov, Mikhail; Dowhan, William; Vitrac, Heidi

    2014-01-01

    Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein Trafficking

  2. Alignment of Helical Membrane Protein Sequences Using AlignMe

    PubMed Central

    Khafizov, Kamil; Forrest, Lucy R.

    2013-01-01

    Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set. PMID:23469223

  3. Comparison of helix interactions in membrane and soluble alpha-bundle proteins.

    PubMed Central

    Eilers, Markus; Patel, Ashish B; Liu, Wei; Smith, Steven O

    2002-01-01

    Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with

  4. Novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

    SciTech Connect

    Nelson, N.

    1987-09-01

    A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of /sup 125/I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of /sup 125/I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with /sup 125/I-labeled alpha-bungarotoxin and antigens with /sup 125/I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the /sup 125/I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.

  5. Fast and efficient protein purification using membrane adsorber systems.

    PubMed

    Suck, Kirstin; Walter, Johanna; Menzel, Frauke; Tappe, Alexander; Kasper, Cornelia; Naumann, Claudia; Zeidler, Robert; Scheper, Thomas

    2006-02-10

    The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  7. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

    SciTech Connect

    Price, E.M.; Freisheim, J.H.

    1987-07-28

    A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.

  8. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

    PubMed Central

    Costello, Shawn M.; Plummer, Ashlee M.; Fleming, Patrick J.; Fleming, Karen G.

    2016-01-01

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

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

    PubMed

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

    2016-08-02

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

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

    PubMed Central

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

    2016-01-01

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

  11. From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein.

    PubMed

    Vos, Werner L; Nazarov, Petr V; Koehorst, Rob B M; Spruijt, Ruud B; Hemminga, Marcus A

    2009-05-01

    The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.

  12. Prediction of membrane protein types using maximum variance projection

    NASA Astrophysics Data System (ADS)

    Wang, Tong; Yang, Jie

    2011-05-01

    Predicting membrane protein types has a positive influence on further biological function analysis. To quickly and efficiently annotate the type of an uncharacterized membrane protein is a challenge. In this work, a system based on maximum variance projection (MVP) is proposed to improve the prediction performance of me