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Sample records for membrane protein crystallization

  1. 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.

  2. 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.

  3. Crystallization of Membrane Proteins by Vapor Diffusion

    PubMed Central

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

    2016-01-01

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

  4. 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

  5. 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.

  6. Membrane protein structures without crystals, by single particle electron cryomicroscopy

    PubMed Central

    Vinothkumar, Kutti R

    2015-01-01

    It is an exciting period in membrane protein structural biology with a number of medically important protein structures determined at a rapid pace. However, two major hurdles still remain in the structural biology of membrane proteins. One is the inability to obtain large amounts of protein for crystallization and the other is the failure to get well-diffracting crystals. With single particle electron cryomicroscopy, both these problems can be overcome and high-resolution structures of membrane proteins and other labile protein complexes can be obtained with very little protein and without the need for crystals. In this review, I highlight recent advances in electron microscopy, detectors and software, which have allowed determination of medium to high-resolution structures of membrane proteins and complexes that have been difficult to study by other structural biological techniques. PMID:26435463

  7. Strategies for crystallization of large membrane protein complexes

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Shinya; Shinzawa-Itoh, Kyoko; Ueda, Hidefumi; Tsukihara, Tomitake; Fukumoto, Yoshihisa; Kubota, Tomomi; Kawamoto, Masahide; Fukuyama, Keiichi; Matsubara, Hiroshi

    1992-08-01

    Crystalline cytochrome c oxidase and ubiquinol: cytochrome c oxidoreductase which diffracted X-rays at 7-8A˚resolution were obtained from bovine heart mitochondria. The methods for the purification and crystallization of these enzymes indicate that large membrane protein complexes are easier to purify and crystallize than smaller homologous membrane protein complexes, because the former have more hydrophilic surface than the latter. Bulky and polydispersed detergents such as Brij-35 and Tween 20 attached to the isolated complex are not always obstructive to crystallization if they are effective for stabilizing the complexes.

  8. An automated pipeline to screen membrane protein 2D crystallization

    PubMed Central

    Kim, Changki; Vink, Martin; Hu, Minghui; Love, James; Stokes, David L.; Ubarretxena-Belandia, Iban

    2011-01-01

    Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility. PMID:20349145

  9. Membrane protein crystallization in lipidic mesophases: detergent effects.

    PubMed Central

    Ai, X; Caffrey, M

    2000-01-01

    The "cubic phase method" for growing crystals of membrane proteins uses a complex mixture of water, lipid, protein, and other components. The current view is that the cubic phase is integral to the process. Thus additives from whatever source introduce the possibility of destabilizing the phase, thereby compromising the crystallization process. Detergents are used to solubilize membrane proteins and are likely to be ported into the cubic medium with the target protein. Depending on the identity and concentration of the detergent, the cubic phase, which itself is membranous, may be solubilized or destabilized in such a way as to render it unsuitable as a crystal growing system. The nonionic detergent n-dodecyl-beta-D-maltopyranoside is commonly used in membrane protein work. In this study, we evaluate its effect on the cubic mesophase of hydrated monoolein. X-ray diffraction was used for phase identification and mesophase microstructure characterization. The results show that while low levels of the detergent are tolerated, increasing concentrations trigger a cubic-to-lamellar phase transition in a temperature-dependent manner. This finding is rationalized in the context of complementary molecular shapes of the lipid and the detergent and has implications for the mechanism of crystallization in lipidic mesophases as discussed. PMID:10866965

  10. Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Porter, Christopher

    2010-01-01

    A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile 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 alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:21113125

  11. Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization

    SciTech Connect

    Perry, S.; Roberts, G; Tice, J; Gennis, R; Kenis, P

    2009-01-01

    We report on a microfluidic method for the formation of aqueous/lipid mesophases to enable screening of suitable crystallization conditions of membrane proteins from a membrane-like phase in sub-20 nL volumes. This integrated microfluidic chip and the employed mixing strategy address the specific challenges associated with the mixing of fluids of highly different viscosities (here a factor of 30) as well as the non-Newtonian character of the resulting mesophases. The chip requires less than 20 nL of material per condition screened, whereas typically on the order of 10 {micro}L or more is needed for a batch preparation in the present screening methods. We validated our approach with the successful crystallization of the membrane protein bacteriorhodopsin.

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

    PubMed Central

    Li, Dianfan; Lee, Jean; Caffrey, Martin

    2011-01-01

    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. PMID:21743796

  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. Detection of Membrane Protein Two-Dimensional Crystals in Living Cells

    PubMed Central

    Gualtieri, E.J.; Guo, F.; Kissick, D.J.; Jose, J.; Kuhn, R.J.; Jiang, W.; Simpson, G.J.

    2011-01-01

    It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation. PMID:21190673

  15. Effects of impurities on membrane-protein crystallization in different systems

    SciTech Connect

    Kors, Christopher A.; Wallace, Ellen; Davies, Douglas R.; Li, Liang; Laible, Philip D.; Nollert, Peter

    2009-10-01

    The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality. If generally applicable, this tolerance may be exploited (i) in initial crystallization trials to determine the ‘crystallizability’ of a given membrane-protein and (ii) to subject partially pure membrane-protein samples to crystallization trials. When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP

  16. Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization

    PubMed Central

    Lasala, R.; Coudray, N.; Abdine, A.; Zhang, Z.; Lopez-Redondo, M.; Kirshenbaum, R.; Alexopoulos, J.; Zolnai, Z.; Stokes, D.L.; Ubarretxena-Belandia, I.

    2014-01-01

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1,300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization. PMID:25478971

  17. Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

    PubMed

    Lasala, R; Coudray, N; Abdine, A; Zhang, Z; Lopez-Redondo, M; Kirshenbaum, R; Alexopoulos, J; Zolnai, Z; Stokes, D L; Ubarretxena-Belandia, I

    2015-02-01

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. 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.

  19. The nanoscience behind the art of in-meso crystallization of membrane proteins.

    PubMed

    Zabara, Alexandru; Meikle, Thomas G; Newman, Janet; Peat, Thomas S; Conn, Charlotte E; Drummond, Calum J

    2017-01-05

    The structural changes occurring at the nanoscale level within the lipid bilayer and driving the in-meso formation of large well-diffracting membrane protein crystals have been uniquely characterized for a model membrane protein, intimin. Importantly, the order to order transitions taking place within the bilayer and the lipidic nanostructures required for crystal growth have been shown to be general, occurring for both the cubic and the sponge mesophase crystallization pathways. For the first time, a transient fluid lamellar phase has been observed and unambiguously assigned for both crystallization pathways, present at the earliest stages of protein crystallogenesis but no longer observed once the crystals surpass the size of the average lyotropic liquid crystalline domain. The reported time-resolved structural investigation provides a significantly improved and general understanding of the nanostructural changes taking place within the mesophase during in-meso crystallization which is a fundamental advance in the enabling area of membrane protein structural biology.

  20. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    SciTech Connect

    Wallace, E.; Dranow, D.; Laible, P. D.; Christensen, J.; Nollert, P.

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  1. Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

    PubMed Central

    Wallace, Ellen; Dranow, David; Laible, Philip D.; Christensen, Jeff; Nollert, Peter

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  2. A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.

    PubMed

    Kolek, Stefan Andrew; Bräuning, Bastian; Stewart, Patrick Douglas Shaw

    2016-04-01

    Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

  3. Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

    PubMed Central

    Kubicek, Jan; Schlesinger, Ramona; Baeken, Christian; Büldt, Georg; Schäfer, Frank; Labahn, Jörg

    2012-01-01

    We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time. PMID:22536388

  4. Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

    SciTech Connect

    D Freed; P Horanyi; M Wiener; D Cafiso

    2011-12-31

    Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B{sub 12}. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

  5. Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

    SciTech Connect

    Freed, Daniel M.; Horanyi, Peter S.; Wiener, Michael C.; Cafiso, David S.

    2010-09-27

    Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B{sub 12}. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

  6. Characterization of New Detergents and Detergent Mimetics by Scattering Techniques for Membrane Protein Crystallization.

    PubMed

    Bonneté, Françoise; Loll, Patrick J

    2017-01-01

    Membrane proteins are difficult to manipulate and stabilize once they have been removed from their native membranes. However, despite these difficulties, successes in membrane-protein structure determination have continued to accumulate for over two decades, thanks to advances in chemistry and technology. Many of these advances have resulted from efforts focused on protein engineering, high-throughput expression, and development of detergent screens, all with the aim of enhancing protein stability for biochemistry and biophysical studies. In contrast, considerably less work has been done to decipher the basic mechanisms that underlie the structure of protein-detergent complexes and to describe the influence of detergent structure on stabilization and crystallization. These questions can be addressed using scattering techniques (employing light, X-rays, and/or neutrons), which are suitable to describe the structure and conformation of macromolecules in solution, as well as to assess weak interactions between particles, both of which are clearly germane to crystallization. These techniques can be used either in batch modes or coupled to size-exclusion chromatography, and offer the potential to describe the conformation of a detergent-solubilized membrane protein and to quantify and model detergent bound to the protein in order to optimize crystal packing. We will describe relevant techniques and present examples of scattering experiments, which allow one to explore interactions between micelles and between membrane protein complexes, and relate these interactions to membrane protein crystallization.

  7. Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

    PubMed Central

    Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2014-01-01

    Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

  8. Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins

    PubMed Central

    Koszelak-Rosenblum, Mary; Krol, Adam; Mozumdar, Namrita; Wunsch, Kristin; Ferin, Adam; Cook, Eleanor; Veatch, Christina K; Nagel, Raymond; Luft, Joseph R; DeTitta, George T; Malkowski, Michael G

    2009-01-01

    Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein-detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye-based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536-cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high-throughput crystallization facility located at the Hauptman-Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X-ray diffraction. PMID:19554626

  9. Crystal structure of Escherichia coli YidC, a membrane protein chaperone and insertase.

    PubMed

    Kumazaki, Kaoru; Kishimoto, Toshiki; Furukawa, Arata; Mori, Hiroyuki; Tanaka, Yoshiki; Dohmae, Naoshi; Ishitani, Ryuichiro; Tsukazaki, Tomoya; Nureki, Osamu

    2014-12-03

    Bacterial YidC, an evolutionally conserved membrane protein, functions as a membrane protein chaperone in cooperation with the Sec translocon and as an independent insertase for membrane proteins. In Gram-negative bacteria, the transmembrane and periplasmic regions of YidC interact with the Sec proteins, forming a multi-protein complex for Sec-dependent membrane protein integration. Here, we report the crystal structure of full-length Escherichia coli YidC. The structure reveals that a hydrophilic groove, formed by five transmembrane helices, is a conserved structural feature of YidC, as compared to the previous YidC structure from Bacillus halodurans, which lacks a periplasmic domain. Structural mapping of the substrate- or Sec protein-contact sites suggested the importance of the groove for the YidC functions as a chaperone and an insertase, and provided structural insight into the multi-protein complex.

  10. 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.

  11. Detergent quantification in membrane protein samples and its application to crystallization experiments.

    PubMed

    Prince, Chelsy C; Jia, Zongchao

    2013-12-01

    The structural characterization of membrane proteins remains a challenging field, largely because the use of stabilizing detergents is required. Researchers must first select a suitable detergent for the solubility and stability of their protein during in vitro studies. In addition, an appropriate concentration of detergent in membrane protein samples can be essential for protein solubility, stability, and experimental success. For example, in membrane protein crystallography, detergent concentration in the crystallization drop can be a critical parameter influencing crystal growth. Over the past decade, multiple techniques have been developed for the measurement of detergent concentration using a wide variety of strategies. These methods include colorimetric reactions, which target specific detergent classes, and analytical techniques applicable to a wide variety of detergents. This review will summarize and discuss the available options. It will be a useful resource to those selecting a strategy that best fits their experimental requirements and available instruments.

  12. Membrane proteins, detergents and crystals: what is the state of the art?

    PubMed

    Loll, Patrick J

    2014-12-01

    At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein-detergent complexes remains the dominant methodology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein-detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors.

  13. Melting of Two-Dimensional Crystals of Membrane-Protein Bacteriorhodopsin.

    NASA Astrophysics Data System (ADS)

    Koltover, I.; Salditt, T.; Rädler, J. O.; Safinya, C. R.; Rothschild, K. J.

    1997-03-01

    Bacteriorhodopsin (bR), a light-driven proton pump, is an integral membrane protein of bacterium Halobacterium Salinarium. In the native bacterial membrane bR self-assembles into regular hexagonal crystalline arrays in the plane of membrane. This two-dimensional (2D) protein crystal undergoes a fully reversible melting transition as a function of temperature. We have conducted a synchrotron x-ray diffraction study of oriented multilayers of bR-containing native bacterial membrane patches and of of novel giant (50μm) single-crystal fused bR membranes. The precise in-situ control of humidity and sample temperature combined with the line-shape analyses allowed us to elucidate and control the protein-protein interactions. The important findings are as follows. First, the ordered 2D self-assembled lattice of proteins exhibits diffraction patterns characteristic of a 2D solid with power-law decay of in-plane positional correlations, which allows to measure the elastic constants of protein crystal. Second, the melting temperature is a function of the multilayer hydration, with two distinct regimes of melting. We discuss the models of interactions in bR multilayers consistent with our data. Supported by NSF grant DMR-962091, and the Petroleum Research Fund (No.31352-AC7).

  14. Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins.

    PubMed

    Rummel; Hardmeyer; Widmer; Chiu; Nollert; Locher; Pedruzzi; Landau; Rosenbusch

    1998-01-01

    The major constraint in attaining high resolution structures of membrane proteins by X-ray crystallography is the growth of well-ordered three-dimensional crystals. To enable such crystallizations, we have used lipidic cubic phases consisting of monoglycerides and water. Bacteriorhodopsin and lysozyme, as paradigms of membrane and soluble proteins, nucleate and grow to well-ordered crystals that diffract X-rays isotropically in all three dimensions to 2.0 Å. We envisage bacteriorhodopsin to partition into, and diffuse within, the bilayer of a lipidic cubic matrix, while the polar lysozyme resides in the aqueous compartment thereof. The phenomenology of bicontinuous cubic phases, consisting of curved bilayers whose structures follow infinitely periodic minimal surfaces (IPMS), is presented. Detailed prescriptions of the preparation of lipidic cubic phase matrices are given and their potential for the crystallization of other biological macromolecules is discussed. Copyright 1998 Academic Press.

  15. 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.

  16. Membrane proteins, detergents and crystals: what is the state of the art?

    PubMed Central

    Loll, Patrick J.

    2014-01-01

    At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein–detergent complexes remains the dominant method­ology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein–detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors. PMID:25484203

  17. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    PubMed Central

    Chae, Pil Seok; Rasmussen, Søren G. F.; Rana, Rohini; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A.; Kruse, Andrew C.; Nurva, Shailika; Loland, Claus J.; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G.; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H.

    2011-01-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family display favorable behavior relative to conventional detergents, as tested on multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied. PMID:21037590

  18. Automated Electron Microscopy for Evaluating Two-dimensional Crystallization of Membrane Proteins

    PubMed Central

    Hu, Minghui; Vink, Martin; Kim, Changki; Derr, KD; Koss, John; D'Amico, Kevin; Cheng, Anchi; Pulokas, James; Ubarretxena-Belandia, Iban; Stokes, David

    2010-01-01

    Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen. PMID:20197095

  19. Two-dimensional crystallization of integral membrane proteins for electron crystallography.

    PubMed

    Stokes, David L; Rice, William J; Hu, Minghui; Kim, Changki; Ubarretxena-Belandia, Iban

    2010-01-01

    Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microscopy.

  20. Two-Dimensional Crystallization of Integral Membrane Proteins for Electron Crystallography

    PubMed Central

    Stokes, David L.; Rice, William J.; Hu, Minghui; Kim, Changki; Ubarretxena, Iban

    2011-01-01

    Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microsopy. PMID:20665267

  1. Electron Cryomicroscopy of Membrane Proteins: Specimen Preparation for Two-Dimensional Crystals and Single Particles

    PubMed Central

    Schmidt-Krey, Ingeborg; Rubinstein, John L.

    2010-01-01

    Membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-EM) methods: electron crystallography of two-dimensional (2D) crystals and single particle analysis of detergent-solubilized protein complexes. To obtain the highest-possible resolution data from membrane proteins, whether prepared as 2D crystals or single particles, cryo-EM samples must be vitrified with great care. Grid preparation for cryo-EM of 2D crystals is possible by back-injection, the carbon sandwich technique, drying in sugars before cooling in the electron microscope, or plunge-freezing. Specimen grids for single particle cryo-EM studies of membrane proteins are usually produced by plunge-freezing protein solutions, supported either by perforated or a continuous carbon film substrate. This review outlines the different techniques available and the suitability of each method for particular samples and studies. Experimental considerations in sample preparation and preservation include the protein itself and the presence of lipid or detergent. The appearance of cryo-EM samples in different conditions is also discussed. PMID:20678942

  2. Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

    PubMed Central

    Lacapère, J J; Stokes, D L; Chatenay, D

    1992-01-01

    We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:1420878

  3. High-throughput crystallization of membrane proteins using the lipidic bicelle method.

    PubMed

    Ujwal, Rachna; Abramson, Jeff

    2012-01-09

    Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent's inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization. Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation. Because

  4. High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

    PubMed Central

    Ujwal, Rachna; Abramson, Jeff

    2012-01-01

    Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent′s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization. Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3

  5. The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.

    PubMed

    Iacovache, Ioan; Biasini, Marco; Kowal, Julia; Kukulski, Wanda; Chami, Mohamed; van der Goot, F Gisou; Engel, Andreas; Rémigy, Hervé-W

    2010-03-01

    Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution.

  6. Structure determination of an integral membrane protein at room temperature from crystals in situ.

    PubMed

    Axford, Danny; Foadi, James; Hu, Nien Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-06-01

    The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  7. Structure determination of an integral membrane protein at room temperature from crystals in situ

    PubMed Central

    Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-01-01

    The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines. PMID:26057664

  8. Crystal structure of a major outer membrane protein from Thermus thermophilus HB27.

    PubMed

    Brosig, Alexander; Nesper, Jutta; Boos, Winfried; Welte, Wolfram; Diederichs, Kay

    2009-02-06

    The thermophilic eubacterium Thermus thermophilus belongs to one of the oldest branches of evolution and has a multilayered cell envelope that differs from that of modern Gram-negative bacteria. Its outer membrane contains integral outer membrane proteins (OMPs), of which only a few are characterized. TtoA, a new beta-barrel OMP, was identified by searching the genome sequence of strain HB27 for the presence of a C-terminal signature sequence. The structure of TtoA was determined to a resolution of 2.8 A, representing the first crystal structure of an OMP from a thermophilic bacterium. TtoA consists of an eight-stranded beta-barrel with a large extracellular part to which a divalent cation is bound. A five-stranded extracellular beta-sheet protrudes out of the membrane-embedded transmembrane barrel and is stabilized by a disulfide bridge. The edge of this beta-sheet forms crystal contacts that could mimic interactions with other proteins. In modern Gram-negative bacteria, the C-terminal signature sequence of OMPs is required for binding to an Omp85 family protein as a prerequisite for its assembly. We present hints that a similar assembly pathway exists in T. thermophilus by an in vitro binding assay, where unfolded TtoA binds to the Thermus Omp85 family protein TtOmp85, while a mutant without the signature sequence does not.

  9. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    SciTech Connect

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-07-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  10. Structure determination of an integral membrane protein at room temperature from crystals in situ

    SciTech Connect

    Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-05-14

    The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  11. High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.

    PubMed

    Gabrielsen, Mads; Kroner, Frank; Black, Isobel; Isaacs, Neil W; Roe, Andrew J; McLuskey, Karen

    2011-01-01

    An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.

  12. Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method

    SciTech Connect

    Li, Dianfan; Pye, Valerie E.; Caffrey, Martin

    2015-01-01

    Very little information is available in the literature concerning the experimental heavy-atom phasing of membrane-protein structures where the crystals have been grown using the lipid cubic phase (in meso) method. In this paper, pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine labelling as applied to an integral membrane kinase crystallized in meso are described. An assay to assess cysteine accessibility for mercury labelling of membrane proteins is introduced. Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.

  13. Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

    PubMed Central

    Li, Liang; Mustafi, Debarshi; Fu, Qiang; Tereshko, Valentina; Chen, Delai L.; Tice, Joshua D.; Ismagilov, Rustem F.

    2006-01-01

    High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a “hybrid” droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as ≈140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in ≈10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, ≈1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization. PMID:17159147

  14. Solution structure of detergent micelles at conditions relevant to membrane protein crystallization.

    SciTech Connect

    Littrell, K.; Thiyagarajan, P.; Tiede, D.; Urban, V.

    1999-07-02

    In this study small angle neutron scattering was used to characterize the formation of micelles in aqueous solutions of the detergents DMG and SPC as a function of detergent concentration and ionic strength of the solvent. The effects on the micelle structure of the additives glycerol and PEG, alone as well as in combination typical for actual membrane protein crystallization, were also explored. This research suggests that the micelles are cigar-like in form at the concentrations studied. The size of the micelles was observed to increase with increasing ionic strength but decrease with the addition of glycerol or PEG.

  15. The funnel approach to the pre-crystallization production of membrane proteins

    PubMed Central

    Lewinson, Oded; Lee, Allen T.; Rees, Douglas C.

    2008-01-01

    SUMMARY Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcome these difficulties, and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs are generated and tested for expression in E. coli, membrane localization, detergent extraction, and homogeneity. High throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and most importantly the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells, to extent of membrane insertion, and even on protein function. Following the initial stream lined screening, we were able to fine tune and produce 9 of the 36 targets as material suitable for crystallization or other structural studies. PMID:18241890

  16. Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

    SciTech Connect

    Su, Chih-Chia; Yang, Feng; Long, Feng; Reyon, Deepak; Routh, Mathew D.; Kuo, Dennis W.; Mokhtari, Adam K.; Van Ornam, Jonathan D.; Rabe, Katherine L.; Hoy, Julie A.; Lee, Young Jin; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

    2010-03-29

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu{sup +} and Ag{sup +} ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly {beta}-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the {alpha}-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first {beta}-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu{sup +} and Ag{sup +} were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

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

    PubMed

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

    2008-12-01

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

  18. Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction.

    PubMed

    Hall, Spencer E; Roberts, Kyle; Vaidehi, Nagarajan

    2009-01-01

    The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i - 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4-14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these

  19. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.

    PubMed

    Li, Dianfan; Boland, Coilín; Aragao, David; Walsh, Kilian; Caffrey, Martin

    2012-09-02

    An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases(1-5), has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field(6-21) (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting(22,23). Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)(24,25) are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies(4,26). The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been

  20. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  1. X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination

    SciTech Connect

    Khvostichenko, Daria S.; Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; Laible, Philip D.; Kenis, Paul J. A.

    2014-10-01

    ABSTRACT: Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting of individual crystals. We validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.

  2. X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination

    DOE PAGES

    Khvostichenko, Daria S.; Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; ...

    2014-08-21

    Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting ofmore » individual crystals. In addition, we validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.« less

  3. Improving protein crystal quality by selective removal of a Ca{sup 2+}-dependent membrane-insertion loop

    SciTech Connect

    Neau, David B.; Gilbert, Nathaniel C.; Bartlett, Sue G.; Dassey, Adam; Newcomer, Marcia E.

    2007-11-01

    Protein engineering dramatically enhances the quality of crystals of a Ca{sup 2+}-dependent membrane-binding protein. Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca{sup 2+}-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 Å crystal structure of 8R-LOX and spectroscopic data suggested that Ca{sup 2+} stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Δ41–45:GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 Å improvement in the resolution of the diffraction data.

  4. Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli.

    PubMed

    Köster, Stefan; van Pee, Katharina; Yildiz, Özkan

    2015-01-01

    OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.

  5. Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy

    PubMed Central

    2015-01-01

    The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.2013, 13, 2846−2857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method. PMID:24803849

  6. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27

    PubMed Central

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-01-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted β-strands. TTC0834 was expressed with an N-­terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 Å resolution and the best diffracting crystals belonged to space group P3121 or P3221, with unit-cell parameters a = b = 166.67, c = 97.53 Å. PMID:18540069

  7. Expression, crystallization and preliminary X-ray analysis of an outer membrane protein from Thermus thermophilus HB27.

    PubMed

    Brosig, Alexander; Nesper, Jutta; Welte, Wolfram; Diederichs, Kay

    2008-06-01

    The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted beta-strands. TTC0834 was expressed with an N-terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 A resolution and the best diffracting crystals belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 166.67, c = 97.53 A.

  8. Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method

    PubMed Central

    Li, Dianfan; Pye, Valerie E.; Caffrey, Martin

    2015-01-01

    Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method. PMID:25615865

  9. Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase

    PubMed Central

    Li, Dianfan; Shah, Syed T. A.; Caffrey, Martin

    2013-01-01

    A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials. PMID:23956688

  10. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

    PubMed Central

    Hill, C P; Worthylake, D; Bancroft, D P; Christensen, A M; Sundquist, W I

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus. Images Fig. 1 Fig. 2 Fig. 3 PMID:8610175

  11. A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser.

    PubMed

    Hato, Masakatsu; Hosaka, Toshiaki; Tanabe, Hiroaki; Kitsunai, Tokuji; Yokoyama, Shigeyuki

    2014-09-01

    A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.

  12. 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.

  13. Structure determination using poorly diffracting membrane-protein crystals: the H+-ATPase and Na+,K+-ATPase case history.

    PubMed

    Pedersen, Bjørn P; Morth, J Preben; Nissen, Poul

    2010-03-01

    An approach is presented for the structure determination of membrane proteins on the basis of poorly diffracting crystals which exploits molecular replacement for heavy-atom site identification at 6-9 A maximum resolution and improvement of the heavy-atom-derived phases by multi-crystal averaging using quasi-isomorphous data sets. The multi-crystal averaging procedure allows real-space density averaging followed by phase combination between non-isomorphous native data sets to exploit crystal-to-crystal nonisomorphism despite the crystals belonging to the same space group. This approach has been used in the structure determination of H(+)-ATPase and Na(+),K(+)-ATPase using Ca(2+)-ATPase models and its successful application to the Mhp1 symporter using LeuT as a search model is demonstrated.

  14. The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

    SciTech Connect

    Hu, Junbin; Li, Jingzhi; Qian, Xinguo; Denic, Vlad; Sha, Bingdong

    2010-08-16

    Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a 'finger' domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an 'open' conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a 'closed' conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.

  15. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

    PubMed

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

    TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

  16. Liquid-liquid diffusion profiles in microgravity experiments, and crystal growth of the membrane protein bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Wagner, Gottfried; Linhardt, Rudolf

    1991-03-01

    Bacteriorhodopsin (BR) is a light-driven electrogenic proton pump from Halobacterium halobium. BR crystals, particularly into the third dimension, are ordered by relatively weak intermolecular bonds, possibly susceptible to disturbing solutal convection. Minimized convection is given under microgravity, e.g., during IML or EURECA space missions, but duration may not be long enough to terminate the crystal growth under microgravity. At the distal end of the PCF protein chamber, kinetics of measured protein crystal growth and of salt/protein diffusion will be demonstrated here: equilibrium of salt diffusion and of BR crystal growth is reached within a time span of 100 to 200 h; similar values are predicted by the CRYSLIB computer program for the CRYOSTAT protein chamber.

  17. A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization.

    PubMed

    Hovers, Jens; Potschies, Meike; Polidori, Ange; Pucci, Bernard; Raynal, Simon; Bonneté, Françoise; Serrano-Vega, Maria J; Tate, Christopher G; Picot, Daniel; Pierre, Yves; Popot, Jean-Luc; Nehmé, Rony; Bidet, Michel; Mus-Veteau, Isabelle; Busskamp, Holger; Jung, Karl-Heinz; Marx, Andreas; Timmins, Peter A; Welte, Wolfram

    2011-04-01

    Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.

  18. Simple Host—Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    PubMed Central

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2008-01-01

    This paper utilizes cyclodextrin-based host—guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-β-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way. PMID:18831551

  19. Simple Host−Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    SciTech Connect

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2009-01-15

    This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-{beta}-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

  20. Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

    PubMed

    Panwar, Pankaj; Deniaud, Aurélien; Pebay-Peyroula, Eva

    2012-10-01

    Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.

  1. A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization

    PubMed Central

    Hovers, Jens; Potschies, Meike; Polidori, Ange; Pucci, Bernard; Raynal, Simon; Bonneté, Françoise; Serrano-Vega, Maria J.; Tate, Christopher G.; Picot, Daniel; Pierre, Yves; Popot, Jean-Luc; Nehmé, Rony; Bidet, Michel; Mus-Veteau, Isabelle; Bußkamp, Holger; Jung, Karl-Heinz; Marx, Andreas; Timmins, Peter A.; Welte, Wolfram

    2013-01-01

    Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside (“C12-b-M”) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors by approximately 10°C compared to C12-b-M. Another surfactant yielded a stabilization of the human Patched protein receptor by 13°C. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. PMID:21314479

  2. Advanced Protein Crystallization Facility (APCF)

    NASA Technical Reports Server (NTRS)

    1998-01-01

    This section of the Life and Microgravity Spacelab (LMS) publication contains articles entitled: (1) Crystallization of EGFR-EGF; (2) Crystallization of Apocrustacyanin C1; (3) Crystallization and X-ray Analysis of 5S rRNA and the 5S rRNA Domain A; (4) Growth of Lysozyme Crystals at Low Nucleation Density; (5) Comparative Analysis of Aspartyl tRNA-synthetase and Thaumatin Crystals Grown on Earth and In Microgravity; (6) Lysosome Crystal Growth in the Advanced Protein Crystallization Facility Monitored via Mach-Zehnder Interferometry and CCD Video; (7) Analysis of Thaumatin Crystals Grown on Earth and in Microgravity; (8) Crystallization of the Nucleosome Core Particle; (9) Crystallization of Photosystem I; (10) Mechanism of Membrane Protein Crystal Growth: Bacteriorhodopsin-mixed Micelle Packing at the Consolution Boundary, Stabilized in Microgravity; (11) Crystallization in a Microgravity Environment of CcdB, a Protein Involved in the Control of Cell Death; and (12) Crystallization of Sulfolobus Solfataricus

  3. A fluorescence-detection size-exclusion chromatography-based thermostability assay to identify membrane protein expression and crystallization conditions

    PubMed Central

    Hattori, Motoyuki; Hibbs, Ryan E.; Gouaux, Eric

    2012-01-01

    SUMMARY Optimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein stability require large amounts of material, and are therefore ill-suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram to microgram amounts of the target protein under a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids. PMID:22884106

  4. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    PubMed

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.

    PubMed

    Rouse, Sarah L; Hawthorne, Wlliam J; Lambert, Sebastian; Morgan, Marc L; Hare, Stephen A; Matthews, Stephen

    2016-12-01

    Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF(106-430) from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.

  6. Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter

    PubMed Central

    Rouse, Sarah L.; Hawthorne, Wlliam J.; Lambert, Sebastian; Morgan, Marc L.; Hare, Stephen A.; Matthews, Stephen

    2016-01-01

    Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF106–430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems. PMID:27917837

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

    PubMed

    Caffrey, Martin

    2015-01-01

    The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published

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

    PubMed Central

    Caffrey, Martin

    2015-01-01

    The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published

  9. Membrane Protein Crystallization in Lipidic Mesophases. Hosting Lipid Effects on the Crystallization and Structure of a Transmembrane Peptide

    SciTech Connect

    Hfer, Nicole; Aragao, David; Lyons, Joseph A.; Caffrey, Martin

    2011-09-28

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating d- and l-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double-stranded, intertwined double helix (DSDH) or as a head-to-head single-stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis, the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesized lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high-quality, structure-grade crystals with gramicidin only in the DSDH conformation.

  10. Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide

    PubMed Central

    Höfer, Nicole; Aragão, David; Lyons, Joseph A.; Caffrey, Martin

    2012-01-01

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation. PMID:22933857

  11. Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide.

    PubMed

    Höfer, Nicole; Aragão, David; Lyons, Joseph A; Caffrey, Martin

    2011-04-06

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation.

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

    SciTech Connect

    Caffrey, Martin

    2015-01-01

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

  14. 2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins.

    PubMed

    Jeckelmann, Jean-Marc; Harder, Daniel; Ucurum, Zöhre; Fotiadis, Dimitrios

    2014-10-01

    Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

  15. The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

    PubMed

    Sorek, Nadav; Sorek, Hagit; Kijac, Aleksandra; Szemenyei, Heidi J; Bauer, Stefan; Hématy, Kian; Wemmer, David E; Somerville, Chris R

    2014-12-12

    Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1-4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1-4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1-4-glucan chains by acting as a "polysaccharide chaperone." © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane*

    PubMed Central

    Sorek, Nadav; Sorek, Hagit; Kijac, Aleksandra; Szemenyei, Heidi J.; Bauer, Stefan; Hématy, Kian; Wemmer, David E.; Somerville, Chris R.

    2014-01-01

    Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.” PMID:25331944

  17. Crystallizing the function of the magnetosome membrane mineralization protein Mms6

    PubMed Central

    Staniland, Sarah S.; Rawlings, Andrea E.

    2016-01-01

    The literature on the magnetosome membrane (MM) protein, magnetosome membrane specific6 (Mms6), is reviewed. Mms6 is native to magnetotactic bacteria (MTB). These bacteria take up iron from solution and biomineralize magnetite nanoparticles within organelles called magnetosomes. Mms6 is a small protein embedded on the interior of the MM and was discovered tightly associated with the formed mineral. It has been the subject of intensive research as it is seen to control the formation of particles both in vivo and in vitro. Here, we compile, review and discuss the research detailing Mms6’s activity within the cell and in a range of chemical in vitro methods where Mms6 has a marked effect on the composition, size and distribution of synthetic particles, with approximately 21 nm in size for solution precipitations and approximately 90 nm for those formed on surfaces. Furthermore, we review and discuss recent work detailing the structure and function of Mms6. From the evidence, we propose a mechanism for its function as a specific magnetite nucleation protein and summaries the key features for this action: namely, self-assembly to display a charged surface for specific iron binding, with the curvature of the surfaces determining the particle size. We suggest these may aid design of biomimetic additives for future green nanoparticle production. PMID:27284056

  18. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

    PubMed

    Warren, Anna J; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R; Horrell, Sam; McAuley, Katherine E; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-07-01

    The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

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

    SciTech Connect

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

    2004-12-17

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

  20. 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

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

    PubMed Central

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

    2015-01-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach. PMID:25849400

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

    PubMed

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

    2015-04-01

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

  3. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    SciTech Connect

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo

    2009-07-31

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  4. Crystal Structure of the Outer Membrane Protein OpdK from Pseudomonas aeruginosa

    SciTech Connect

    Biswas,S.; Mohammad, M.; Movileanu, L.; van den Berg, B.

    2008-01-01

    In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded {beta} barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel ({approx}8 Angstroms diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side--features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.

  5. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  6. Crystallization and preliminary X-ray diffraction analysis of YidC, a membrane-protein chaperone and insertase from Bacillus halodurans

    SciTech Connect

    Kumazaki, Kaoru; Tsukazaki, Tomoya; Nishizawa, Tomohiro; Tanaka, Yoshiki; Kato, Hideaki E.; Nakada-Nakura, Yoshiko; Hirata, Kunio; Mori, Yoshihiro; Suga, Hiroaki; Dohmae, Naoshi; Ishitani, Ryuichiro; Nureki, Osamu

    2014-07-23

    YidC, a membrane-protein chaperone/insertase from B. halodurans, was expressed, purified and crystallized in the lipidic cubic phase. An X-ray diffraction data set was collected to 2.4 Å resolution. YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

  7. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  8. The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A resolution.

    PubMed

    Federici, Luca; Du, Dijun; Walas, Fabien; Matsumura, Hiroyoshi; Fernandez-Recio, Juan; McKeegan, Kenneth S; Borges-Walmsley, M Ines; Luisi, Ben F; Walmsley, Adrian R

    2005-04-15

    Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

  9. Single Crystal Membranes

    NASA Technical Reports Server (NTRS)

    Stormont, R. W.; Morrison, A.

    1974-01-01

    Single crystal a- and c-axis tubes and ribbons of sodium beta-alumina and sodium magnesium beta-alumina were grown from sodium oxide rich melts. Additional experiments grew ribbon crystals containing sodium magnesium beta, beta double prime, beta triple prime, and beta quadruple prime. A high pressure crystal growth chamber, sodium oxide rich melts, and iridium for all surfaces in contact with the melt were combined with the edge-defined, film-fed growth technique to grow the single crystal beta-alumina tubes and ribbons. The crystals were characterized using metallographic and X-ray diffraction techniques, and wet chemical analysis was used to determine the sodium, magnesium, and aluminum content of the grown crystals.

  10. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  11. Microgravity protein crystallization

    PubMed Central

    McPherson, Alexander; DeLucas, Lawrence James

    2015-01-01

    Over the past 20 years a variety of technological advances in X-ray crystallography have shortened the time required to determine the structures of large macromolecules (i.e., proteins and nucleic acids) from several years to several weeks or days. However, one of the remaining challenges is the ability to produce diffraction-quality crystals suitable for a detailed structural analysis. Although the development of automated crystallization systems combined with protein engineering (site-directed mutagenesis to enhance protein solubility and crystallization) have improved crystallization success rates, there remain hundreds of proteins that either cannot be crystallized or yield crystals of insufficient quality to support X-ray structure determination. In an attempt to address this bottleneck, an international group of scientists has explored use of a microgravity environment to crystallize macromolecules. This paper summarizes the history of this international initiative along with a description of some of the flight hardware systems and crystallization results. PMID:28725714

  12. A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data

    PubMed Central

    Dilanian, R A; Darmanin, C; Varghese, J N; Wilkins, S W; Oka, T; Yagi, N; Quiney, H M; Nugent, K A

    2011-01-01

    The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X-ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low-resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two-dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low-resolution phase information that can be refined further by the conventional methods of protein crystallography. PMID:21154412

  13. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  14. General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays.

    PubMed

    Tomasiak, Thomas M; Pedersen, Bjørn P; Chaudhary, Sarika; Rodriguez, Andrew; Colmanares, Yaneth Robles; Roe-Zurz, Zygy; Thamminana, Sobha; Tessema, Meseret; Stroud, Robert M

    2014-08-01

    This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.

  15. How To Minimize Artifacts in Atomistic Simulations of Membrane Proteins, Whose Crystal Structure Is Heavily Engineered: β₂-Adrenergic Receptor in the Spotlight.

    PubMed

    Manna, Moutusi; Kulig, Waldemar; Javanainen, Matti; Tynkkynen, Joona; Hensen, Ulf; Müller, Daniel J; Rog, Tomasz; Vattulainen, Ilpo

    2015-07-14

    Atomistic molecular dynamics (MD) simulations are used extensively to elucidate membrane protein properties. These simulations are based on three-dimensional protein structures that in turn are often based on crystallography. The protein structures resolved in crystallographic studies typically do not correspond to pristine proteins, however. Instead the crystallized proteins are commonly engineered, including structural modifications (mutations, replacement of protein sequences by antibodies, bound ligands, etc.) whose impact on protein structure and dynamics is largely unknown. Here we explore this issue through atomistic MD simulations (∼5 μs in total), focusing on the β2-adrenergic receptor (β2AR) that is one of the most studied members of the G-protein coupled receptor superfamily. Starting from an inactive-state crystal structure of β2AR, we remove the many modifications in β2AR systematically one at a time, in six consecutive steps. After each step, we equilibrate the system and simulate it quite extensively. The results of this step-by-step approach highlight that the structural modifications used in crystallization can affect ligand and G-protein binding sites, packing at the transmembrane-helix interface region, and the dynamics of connecting loops in β2AR. When the results of the systematic step-by-step approach are compared to an all-at-once technique where all modifications done on β2AR are removed instantaneously at the same time, it turns out that the step-by-step method provides results that are superior in terms of maintaining protein structural stability. The results provide compelling evidence that for membrane proteins whose 3D structure is based on structural engineering, the preparation of protein structure for atomistic MD simulations is a delicate and sensitive process. The results show that most valid results are found when the structural modifications are reverted slowly, one at a time.

  16. Dynamic pictures of membrane proteins in two-dimensional crystal, lipid bilayer and detergent as revealed by site-directed solid-state 13C NMR.

    PubMed

    Saitô, Hazime

    2004-11-01

    We have compared site-directed 13C solid-state NMR spectra of [3-13C]Ala- and/or [1-13C]Val-labeled membrane proteins, including bacteriorhodopsin (bR), pharaonis phoborhodopin (ppR), its cognate transducer (pHtrII) and Escherichia coli diacylglycerol kinase (DGK), in two-dimensional (2D) crystal, lipid bilayers, and detergent. Restricted fluctuation motions of these membrane proteins due to oligomerization of bR by specific protein-protein interactions in the 2D crystalline lattice or protein complex between ppR and pHtrII provide the most favorable environment to yield well-resolved, fully visible 13C NMR signals for [3-13C]Ala-labeled proteins. In contrast, several signals from such membrane proteins were broadened or lost owing to interference of inherent fluctuation frequencies (10(4)-10(5)Hz) with frequency of either proton decoupling or magic angle spinning, if their 13C NMR spectra were recorded as a monomer in lipid bilayers at ambient temperature. The presence of such protein dynamics is essential for the respective proteins to achieve their own biological functions. Finally, spectral broadening found for bR and DGK in detergents were discussed.

  17. A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

    PubMed Central

    2016-01-01

    In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine. PMID:28261000

  18. A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals.

    PubMed

    Broecker, Jana; Klingel, Viviane; Ou, Wei-Lin; Balo, Aidin R; Kissick, David J; Ogata, Craig M; Kuo, Anling; Ernst, Oliver P

    2016-11-02

    In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

  19. A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

    SciTech Connect

    Broecker, Jana; Klingel, Viviane; Ou, Wei-Lin; Balo, Aidin R.; Kissick, David J.; Ogata, Craig M.; Kuo, Anling; Ernst, Oliver P.

    2016-10-12

    In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometerbased X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

  20. There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization.

    PubMed

    Veesler, David; Blangy, Stéphanie; Cambillau, Christian; Sciara, Giuliano

    2008-10-01

    In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 514.0 A. After molecular-replacement attempts using available CorA structures as search models failed to yield a solution, it was discovered that the crystals consisted of an Escherichia coli contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination.

  1. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  2. Process for Encapsulating Protein Crystals

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Mosier, Benjamin

    2003-01-01

    A process for growing protein crystals encapsulated within membranes has been invented. This process begins with the encapsulation of a nearly saturated aqueous protein solution inside semipermeable membranes to form microcapsules. The encapsulation is effected by use of special formulations of a dissolved protein and a surfactant in an aqueous first liquid phase, which is placed into contact with a second, immiscible liquid phase that contains one or more polymers that are insoluble in the first phase. The second phase becomes formed into the semipermeable membranes that surround microglobules of the first phase, thereby forming the microcapsules. Once formed, the microcapsules are then dehydrated osmotically by exposure to a concentrated salt or polymer solution. The dehydration forms supersaturated solutions inside the microcapsules, thereby enabling nucleation and growth of protein crystals inside the microcapsules. By suitable formulation of the polymer or salt solution and of other physical and chemical parameters, one can control the rate of transport of water out of the microcapsules through the membranes and thereby create physicochemical conditions that favor the growth, within each microcapsule, of one or a few large crystals suitable for analysis by x-ray diffraction. The membrane polymer can be formulated to consist of low-molecular-weight molecules that do not interfere with the x-ray diffraction analysis of the encapsulated crystals. During dehydration, an electrostatic field can be applied to exert additional control over the rate of dehydration. This protein-crystal-encapsulation process is expected to constitute the basis of protein-growth experiments to be performed on the space shuttle and the International Space Station. As envisioned, the experiments would involve the exposure of immiscible liquids to each other in sequences of steps under microgravitational conditions. The experiments are expected to contribute to knowledge of the precise

  3. 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

  4. 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

  5. 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.

  6. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  7. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  8. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  9. Laterally Mobile, Functionalized Self-Assembled Monolayers at the Fluorous−Aqueous Interface in a Plug-Based Microfluidic System: Characterization and Testing with Membrane Protein Crystallization

    SciTech Connect

    Kreutz, Jason E.; Li, Liang; Roach, L. Spencer; Hatakeyama, Takuji; Ismagilov, Rustem F.

    2009-11-04

    This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous-aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.

  10. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  11. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  12. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    PubMed

    Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

    2016-03-01

    The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.

  13. Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    2003-01-01

    In order to rapidly and efficiently grow crystals, tools were needed to automatically identify and analyze the growing process of protein crystals. To meet this need, Diversified Scientific, Inc. (DSI), with the support of a Small Business Innovation Research (SBIR) contract from NASA s Marshall Space Flight Center, developed CrystalScore(trademark), the first automated image acquisition, analysis, and archiving system designed specifically for the macromolecular crystal growing community. It offers automated hardware control, image and data archiving, image processing, a searchable database, and surface plotting of experimental data. CrystalScore is currently being used by numerous pharmaceutical companies and academic and nonprofit research centers. DSI, located in Birmingham, Alabama, was awarded the patent Method for acquiring, storing, and analyzing crystal images on March 4, 2003. Another DSI product made possible by Marshall SBIR funding is VaporPro(trademark), a unique, comprehensive system that allows for the automated control of vapor diffusion for crystallization experiments.

  14. Using Inorganic Crystals To Grow Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.; Mcpherson, Alexander A.

    1989-01-01

    Solid materials serve as nucleating agents. Protein crystals induced by heterogeneous nucleation and in some cases by epitaxy to grow at lower supersaturations than needed for spontaneous nucleation. Heterogeneous nucleation makes possible to grow large, defect-free single crystals of protein more readily. Such protein crystals benefits research in biochemistry and pharmacology.

  15. Using Inorganic Crystals To Grow Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.; Mcpherson, Alexander A.

    1989-01-01

    Solid materials serve as nucleating agents. Protein crystals induced by heterogeneous nucleation and in some cases by epitaxy to grow at lower supersaturations than needed for spontaneous nucleation. Heterogeneous nucleation makes possible to grow large, defect-free single crystals of protein more readily. Such protein crystals benefits research in biochemistry and pharmacology.

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

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

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

  17. Crystal structure of protein Z–dependent inhibitor complex shows how protein Z functions as a cofactor in the membrane inhibition of factor X

    PubMed Central

    Wei, Zhenquan; Yan, Yahui; Carrell, Robin W.

    2009-01-01

    Protein Z (PZ) binds to PZ-dependent inhibitor (ZPI) and accelerates the inhibition of the coagulation protease, activated factor X (FXa), in the presence of phospholipids and Ca2+. A 2.3Å resolution crystal structure of PZ complexed with ZPI shows that ZPI is a typical serine protease inhibitor and that PZ has a serine protease fold with distorted oxyanion hole and S1 pocket. The 2 molecules bind with fully complementary surfaces spanning over 2400Å2 and involving extensive ionic and hydrophobic interactions. ZPI has an unusual shutter region with a negatively charged residue buried within the hydrophobic core of the molecule. This unique Asp213 is critical in maintaining the balanced metastability required for optimal protease inhibition, especially when PZ is bound, with its replacement with Asn resulting in increased thermal stability, but decreased efficiency of protease inhibition. The structure of ZPI shows negatively and positively charged surfaces on top of the molecule, in keeping with mutagenesis studies in this work indicating exosite interactions with FXa when it docks on top of ZPI. As modeled in this study, the γ-carboxy-glutamic acid-containing domains of PZ and FXa enable them to bind to the same phospholipid surfaces on platelet and other membranes, with optimal proximity for the inhibition of FXa by the complexed ZPI. PMID:19528533

  18. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  19. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  20. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  1. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  2. 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.

  3. 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

  4. 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.

  5. Evolutionary origins of membrane proteins

    NASA Astrophysics Data System (ADS)

    Mulkidjanian, Armen Y.; Galperin, Michael Y.

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

  6. Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

    SciTech Connect

    Nayak, V.; Dessau, M; Kucera, K; Anthony, K; Ledizet, M; Modis, Y

    2009-01-01

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  7. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  8. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  9. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Smith, Craig D.; Smith, H. Wilson; Vijay-Kumar, Senadhi; Senadhi, Shobha E.; Ealick, Steven E.; Carter, Daniel C.; Snyder, Robert S.

    1989-01-01

    The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. Space Shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

  10. Introduction to protein crystallization.

    PubMed

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  11. Introduction to protein crystallization

    PubMed Central

    McPherson, Alexander; Gavira, Jose A.

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid–liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies. PMID:24419610

  12. Design, Synthesis and Properties of Branch-chained Maltoside Detergents for Stabilization and Crystallization of Integral Membrane Proteins: Human Connexin 26

    PubMed Central

    Hong, Wen-Xu; Baker, Kent A.; Ma, Xingquan; Stevens, Raymond C.; Yeager, Mark; Zhang, Qinghai

    2010-01-01

    A challenging requirement for structural studies of integral membrane proteins (IMPs) is the use of amphiphiles that replicate the hydrophobic environment of membranes. Progress has been impeded by the limited number of useful detergents and the need for a deeper understanding of their structure-activity relationships. To this end, we designed a family of detergents containing short, branched alkyl chains at the interface between the polar head and apolar tail. This design mimics the second aliphatic chain of lipid molecules and reduces water penetration, thereby increasing the hydrophobicity within the interior of the micelle. To compare with the popular straight-chained maltoside detergents, the branch-chained β-D-maltosides were synthesized efficiently in pure anomeric form. The branch-chained maltosides form smaller micelles by having shorter main chains, while having comparable hydrophobicity to the detergents with only straight chains. Selected branch-chained and straight-chained maltoside detergents were examined for their ability to solubilize, stabilize, and aid the crystallization of human connexin 26, an α-helical IMP that forms hexamers. We showed that the branch-chained maltosides performed as well as straight-chained analogues and enabled crystallization in different space groups. PMID:20232919

  13. Design, synthesis, and properties of branch-chained maltoside detergents for stabilization and crystallization of integral membrane proteins: human connexin 26.

    PubMed

    Hong, Wen-Xu; Baker, Kent A; Ma, Xingquan; Stevens, Raymond C; Yeager, Mark; Zhang, Qinghai

    2010-06-01

    A challenging requirement for structural studies of integral membrane proteins (IMPs) is the use of amphiphiles that replicate the hydrophobic environment of membranes. Progress has been impeded by the limited number of useful detergents and the need for a deeper understanding of their structure-activity relationships. To this end, we designed a family of detergents containing short, branched alkyl chains at the interface between the polar head and the apolar tail. This design mimics the second aliphatic chain of lipid molecules and reduces water penetration, thereby increasing the hydrophobicity within the interior of the micelle. To compare with the popular straight-chained maltoside detergents, the branch-chained beta-D-maltosides were synthesized efficiently in pure anomeric form. The branch-chained maltosides form smaller micelles by having shorter main chains, while having comparable hydrophobicity to the detergents with only straight chains. Selected branch-chained and straight-chained maltoside detergents were examined for their ability to solubilize, stabilize, and aid the crystallization of human connexin 26, an alpha-helical IMP that forms hexamers. We showed that the branch-chained maltosides with optimized micellar properties performed as well as or better than the straight-chained analogues and enabled crystallization in different space groups.

  14. Tracking membrane protein association in model membranes.

    PubMed

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

    2009-01-01

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

  15. 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

  16. Membrane fission by protein crowding.

    PubMed

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

    2017-04-18

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

  17. Insecticidal crystal proteins of Bacillus thuringiensis.

    PubMed Central

    Höfte, H; Whiteley, H R

    1989-01-01

    A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage. Images PMID:2666844

  18. 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.

  19. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Rosenblum, William M.; Delucas, Lawrence J.; Wilson, William W.

    1989-01-01

    Major advances have been made in several of the experimental aspects of protein crystallography, leaving protein crystallization as one of the few remaining bottlenecks. As a result, it has become important that the science of protein crystal growth is better understood and that improved methods for protein crystallization are developed. Preliminary experiments with both small molecules and proteins indicate that microgravity may beneficially affect crystal growth. For this reason, a series of protein crystal growth experiments using the Space Shuttle was initiated. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth. Various optical techniques are being utilized to monitor the crystal growth process from the incipient or nucleation stage and throughout the growth phase. The eventual goal of these studies is to develop a system which utilizes optical monitoring for dynamic control of the crystallization process.

  20. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  1. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  2. Protein Crystal Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.

  3. Path to protein crystallization

    SciTech Connect

    2010-01-01

    Growth of two-dimensional S-layer crystals on supported lipid bilayers observed in solution using in situ atomic force microscopy. This movie shows proteins sticking onto the supported lipid bilayer, forming a mobile phase that condenses into amorphous clusters, and undergoing a phase transition to crystalline clusters composed of 2 to 15 tetramers. These initial clusters then enter a growth phase in which new tetramers form exclusively at unoccupied lattice sites along the cluster edges.

  4. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  5. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  6. Conformation of Membrane Proteins: Bacteriorhodopsin

    DTIC Science & Technology

    1994-05-13

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

  7. Protein Crystal Growth Apparatus for Microgravity

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor); Dowling, Timothy E. (Inventor)

    1997-01-01

    Apparatus for growing protein crystals under microgravity environment includes a plurality of protein growth assemblies stacked one above the other within a canister. Each of the protein growth assemblies includes a tray having a number of spaced apart growth chambers recessed below an upper surface. the growth chambers each having an upstanding pedestal and an annular reservoir about the pedestal for receiving a wick and precipitating agents. A well is recessed below the top of each pedestal to define a protein crystal growth receptacle. A flexible membrane is positioned on the upper surface of each tray and a sealing plate is positioned above each membrane, each sealing plate having a number of bumpers corresponding in number and alignment to the pedestals for forcing the membrane selectively against the upper end of the respective pedestal to seal the reservoir and the receptacle when the sealing plate is forced down.

  8. Membrane Protein Structure Determination in Membrana

    PubMed Central

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

    2014-01-01

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

  9. Membrane protein structure determination in membrana.

    PubMed

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

    2013-09-17

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

  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. Crystallization of Proteins from Crude Bovine Rod Outer Segments☆

    PubMed Central

    Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

  12. 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.

  13. 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.

  14. Development of single crystal membranes

    NASA Technical Reports Server (NTRS)

    Stormont, R. W.; Cocks, F. H.

    1972-01-01

    The design and construction of a high pressure crystal growth chamber was accomplished which would allow the growth of crystals under inert gas pressures of 2 MN/sq m (300 psi). A novel crystal growth technique called EFG was used to grow tubes and rods of the hollandite compounds, BaMgTi7O16, K2MgTi7O16, and tubes of sodium beta-alumina, sodium magnesium-alumina, and potassium beta-alumina. Rods and tubes grown are characterized using metallographic and X-ray diffraction techniques. The hollandite compounds are found to be two or three-phase, composed of coarse grained orientated crystallites. Single crystal c-axis tubes of sodium beta-alumina were grown from melts containing excess sodium oxide. Additional experiments demonstrated that crystals of magnesia doped beta-alumina and potassium beta-alumina also can be achieved by this EFG technique.

  15. Protein Crystals Grown in Space

    NASA Technical Reports Server (NTRS)

    2000-01-01

    A collage of protein and virus crystals, many of which were grown on the U.S. Space Shuttle or Russian Space Station, Mir. The crystals include the proteins canavalin; mouse monoclonal antibody; a sweet protein, thaumatin; and a fungal protease. Viruses are represented here by crystals of turnip yellow mosaic virus and satellite tobacco mosaic virus. The crystals are photographed under polarized light (thus causing the colors) and range in size from a few hundred microns in edge length up to more than a millimeter. All the crystals are grown from aqueous solutions and are useful for X-ray diffraction analysis. Credit: Dr. Alex McPherson, University of California, Irvine.

  16. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Bugg, Charles E.

    1991-01-01

    Studies of protein crystal growth in the microgravity environment in space are described with special attention given to the crystal growth facilities and the techniques used in Space Shuttle experiments. The properties of large space-grown crystals of gamma interferon, elastase, lathyros ochrus lectin I, and few other proteins grown on various STS flights are described. A comparison of the microgravity-grown crystals with the bast earth-grown crystals demonstrated that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts. When crystallization conditions were optimized, the microgravity-grown protein crystals were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolution than their earth-grown counterparts.

  17. Protein crystals and their growth

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2003-01-01

    Recent results on the associations between protein molecules in crystal lattices, crystal-solution surface energy, elastic properties, strength, and spontaneous crystal cracking are reviewed and discussed. In addition, some basic approaches to understanding the solubility of proteins are followed by an overview of crystal nucleation and growth. It is argued that variability of mixing in batch crystallization may be a source of the variation in the number of crystals ultimately appearing in the sample. The frequency at which new molecules join a crystal lattice is measured by the kinetic coefficient and is related to the observed crystal growth rate. Numerical criteria used to discriminate diffusion- and kinetic-limited growth are discussed on this basis. Finally, the creation of defects is discussed with an emphasis on the role of impurities and convection on macromolecular crystal perfection.

  18. Protein Crystals and their Growth

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2004-01-01

    Recent results on binding between protein molecules in crystal lattice, crystal-solution surface energy, elastic properties and strength and spontaneous crystal cracking are reviewed and discussed in the first half of this paper (Sea 2-4). In the second par&, some basic approaches to solubility of proteins are followed by overview on crystal nucleation and growth (Sec 5). It is argued that variability of mixing in batch crystallization may be a source for scattering of crystal number ultimately appearing in the batch. Frequency at which new molecules join crystal lattice is measured by kinetic coefficient and related to the observable crystal growth rate. Numerical criteria to discriminate diffusion and kinetic limited growth are discussed on this basis in Sec 7. In Sec 8, creation of defects is discussed with the emphasis on the role of impurities and convection on macromolecular crystal I;erfection.

  19. Protein crystals and their growth

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2003-01-01

    Recent results on the associations between protein molecules in crystal lattices, crystal-solution surface energy, elastic properties, strength, and spontaneous crystal cracking are reviewed and discussed. In addition, some basic approaches to understanding the solubility of proteins are followed by an overview of crystal nucleation and growth. It is argued that variability of mixing in batch crystallization may be a source of the variation in the number of crystals ultimately appearing in the sample. The frequency at which new molecules join a crystal lattice is measured by the kinetic coefficient and is related to the observed crystal growth rate. Numerical criteria used to discriminate diffusion- and kinetic-limited growth are discussed on this basis. Finally, the creation of defects is discussed with an emphasis on the role of impurities and convection on macromolecular crystal perfection.

  20. Protein Crystals and their Growth

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2004-01-01

    Recent results on binding between protein molecules in crystal lattice, crystal-solution surface energy, elastic properties and strength and spontaneous crystal cracking are reviewed and discussed in the first half of this paper (Sea 2-4). In the second par&, some basic approaches to solubility of proteins are followed by overview on crystal nucleation and growth (Sec 5). It is argued that variability of mixing in batch crystallization may be a source for scattering of crystal number ultimately appearing in the batch. Frequency at which new molecules join crystal lattice is measured by kinetic coefficient and related to the observable crystal growth rate. Numerical criteria to discriminate diffusion and kinetic limited growth are discussed on this basis in Sec 7. In Sec 8, creation of defects is discussed with the emphasis on the role of impurities and convection on macromolecular crystal I;erfection.

  1. 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.

  2. 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.

  3. 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

  4. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  5. 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.

  6. Protein crystal growth tray assembly

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor); Miller, Teresa Y. (Inventor)

    1992-01-01

    A protein crystal growth tray assembly includes a tray that has a plurality of individual crystal growth chambers. Each chamber has a movable pedestal which carries a protein crystal growth compartment at an upper end. The several pedestals for each tray assembly are ganged together for concurrent movement so that the solutions in the various pedestal growth compartments can be separated from the solutions in the tray's growth chambers until the experiment is to be activated.

  7. Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation.

    PubMed

    Qutub, Yasser; Reviakine, Ilya; Maxwell, Carrie; Navarro, Javier; Landau, Ehud M; Vekilov, Peter G

    2004-11-05

    Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.

  8. High density protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rouleau, Robyn (Inventor); Delucas, Lawrence (Inventor); Hedden, Douglas Keith (Inventor)

    2004-01-01

    A protein crystal growth assembly including a crystal growth cell and further including a cell body having a top side and a bottom side and a first aperture defined therethrough, the cell body having opposing first and second sides and a second aperture defined therethrough. A cell barrel is disposed within the cell body, the cell barrel defining a cavity alignable with the first aperture of the cell body, the cell barrel being rotatable within the second aperture. A reservoir is coupled to the bottom side of the cell body and a cap having a top side is disposed on the top side of the cell body. The protein crystal growth assembly may be employed in methods including vapor diffusion crystallization, liquid to liquid crystallization, batch crystallization, and temperature induction batch mode crystallization.

  9. Lasing from fluorescent protein crystals.

    PubMed

    Oh, Heon Jeong; Gather, Malte C; Song, Ji-Joon; Yun, Seok Hyun

    2014-12-15

    We investigated fluorescent protein crystals for potential photonic applications, for the first time to our knowledge. Rod-shaped crystals of enhanced green fluorescent protein (EGFP) were synthesized, with diameters of 0.5-2 μm and lengths of 100-200 μm. The crystals exhibit minimal light scattering due to their ordered structure and generate substantially higher fluorescence intensity than EGFP or dye molecules in solutions. The magnitude of concentration quenching in EGFP crystals was measured to be about 7-10 dB. Upon optical pumping at 485 nm, individual EGFP crystals located between dichroic mirrors generated laser emission with a single-mode spectral line at 513 nm. Our results demonstrate the potential of protein crystals as novel optical elements for self-assembled, micro- or nano-lasers and amplifiers in aqueous environment.

  10. Surface Relaxation in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

    2002-01-01

    Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

  11. Surface Relaxation in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

    2002-01-01

    Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

  12. Protein crystallization apparatus for microgravity

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The Protein Crystallization for Microgravity (DCAM) was developed at NASA's Marshall Space Flight Center. A droplet of solution with protein molecules dissolved in it is isolated in the center of a small well. In orbit, an elastomer seal is lifted so the solution can evaporate and be absorbed by a wick material. This raises the concentration of the solution, thus prompting protein molecules in the solution to form crystals. The principal investigator is Dr. Dan Carter of New Century Pharmaceuticals in Huntsville, AL.

  13. Protein Crystallization Apparatus for Microgravity

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The Protein Crystallization for Microgravity (DCAM) was developed at NASA's Marshall Space Flight Center. A droplet of solution with protein molecules dissolved in it is isolated in the center of a small well. In orbit, an elastomer seal is lifted so the solution can evaporate and be absorbed by a wick material. This raises the concentration of the solution, thus prompting protein molecules in the solution to form crystals. The principal investigator is Dr. Dan Carter of New Century Pharmaceuticals in Huntsville, AL.

  14. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  15. Protein Crystallization Apparatus for Microgravity

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The Protein Crystallization for Microgravity (DCAM) was developed at NASA's Marshall Space Flight Center. A droplet of solution with protein molecules dissolved in it is isolated in the center of a small well. In orbit, an elastomer seal is lifted so the solution can evaporate and be absorbed by a wick material. This raises the concentration of the solution, thus prompting protein molecules in the solution to form crystals. The principal investigator is Dr. Dan Carter of New Century Pharmaceuticals in Huntsville, AL.

  16. Protein crystallization apparatus for microgravity

    NASA Technical Reports Server (NTRS)

    2001-01-01

    The Protein Crystallization for Microgravity (DCAM) was developed at NASA's Marshall Space Flight Center. A droplet of solution with protein molecules dissolved in it is isolated in the center of a small well. In orbit, an elastomer seal is lifted so the solution can evaporate and be absorbed by a wick material. This raises the concentration of the solution, thus prompting protein molecules in the solution to form crystals. The principal investigator is Dr. Dan Carter of New Century Pharmaceuticals in Huntsville, AL.

  17. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  18. 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.

  19. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Carter, Daniel

    1992-01-01

    The overall scientific goals and rationale for growing protein crystals in microgravity are discussed. Data on the growth of human serum albumin crystals which were produced during the First International Microgravity Laboratory (IML-1) are presented. Potential scientific advantages of the utilization of Space Station Freedom are discussed.

  20. 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.

  1. Secondary and lyotropic liquid crystal membranes for improved aqueous separations

    NASA Astrophysics Data System (ADS)

    Nemade, Parag Ramesh

    An effective membrane separation process should have high flux (i.e., volume filtered per unit membrane surface area per unit time) and selectivity (i.e., passage of the desired species and rejection of undesired species). This dissertation examined two approaches, secondary membranes and lyotropic liquid crystal membranes, for improving flux and selectivity in aqueous liquid separations. The first part of my work emphasizes the use of pre-deposited secondary membranes and backflushing for controlling membrane fouling in microfiltration and ultrafiltration of biological mixtures. Use of secondary membranes increased the permeate flux in microfiltration by several fold. Protein transmission is also enhanced due to the presence of the secondary membrane, and the amount of protein recovered is more than twice that obtained during filtration of protein-only solutions under otherwise identical conditions. In ultrafiltration, the flux enhancement due to secondary membranes is 50%, or less. For the second part of my research, I developed and evaluated polymerized lyotropic liquid crystal (LLC) thin-film composite membranes. LLC assemblies provide an opportunity to make nanoporous polymer membranes with precise control over chemical and structural features on the nanometer scale, which is currently lacking in commercial reverse osmosis (RO) and nanofiltration (NF) membranes available today. These LLC composite membranes are prepared by photopolymerization of solution-cast films of LLC monomer on an ultrafiltration support membrane. These LLC membranes appeared to exhibit almost linearly increasing ionic rejection based on ionic diameter. LLC monomer was modified to achieve a 15% reduction in channel diameter, through the use of a larger multivalent Eu3+ cation as the carboxylate counterion. However, the monomers synthesized required use of solvents such as tetrahydrofuran, which resulted in the dissolution and damage of the support membranes used. Therefore, this direction

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

    PubMed

    Arnold, Thomas; Linke, Dirk

    2007-10-01

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

  3. Current trends in protein crystallization.

    PubMed

    Gavira, José A

    2016-07-15

    Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fields trying to shed some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization from a regular-laboratory point of view. It is also devoted to highlight the latest developments and achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput advances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to gather structural information from single particles such as Cryo-EM are also discussed. Finally, current approaches showing the convergence of different structural biology techniques and the cross-methodologies adaptation to tackle the most difficult problems, are presented. Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies applicable at laboratory scale. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Methods for synthesizing microporous crystals and microporous crystal membranes

    DOEpatents

    Dutta, Prabir; Severance, Michael; Sun, Chenhu

    2017-02-07

    A method of making a microporous crystal material, comprising: a. forming a mixture comprising NaOH, water, and one or more of an aluminum source, a silicon source, and a phosphate source, whereupon the mixture forms a gel; b. heating the gel for a first time period, whereupon a first volume of water is removed from the gel and micoroporous crystal nuclei form, the nuclei having a framework; and c.(if a membrane is to be formed) applying the gel to a solid support seeded with microporous crystals having a framework that is the same as the framework of the nuclei; d. heating the gel for a second time period. during which a second volume of water is added to the gel; wherein the rate of addition of the second volume of water is between about 0.5 and about 2.0 fold the rate of removal of the first volume of water.

  5. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

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

  6. 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.

  7. 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

  8. 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.

  9. Protein crystal nucleation in pores

    PubMed Central

    Nanev, Christo N.; Saridakis, Emmanuel; Chayen, Naomi E.

    2017-01-01

    The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials. PMID:28091515

  10. Nucleation precursors in protein crystallization

    PubMed Central

    Vekilov, Peter G.; Vorontsova, Maria A.

    2014-01-01

    Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10−7 to 10−3 of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics. PMID:24598910

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

    PubMed Central

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

    2008-01-01

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

  12. SNARE proteins and 'membrane rafts'.

    PubMed

    Lang, Thorsten

    2007-12-15

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

  13. Observable Protein Crystal Growth Apparatus

    NASA Technical Reports Server (NTRS)

    2001-01-01

    This diagram shows a cross sectrion of the fluid volume of an individual cell in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

  14. Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes*

    PubMed Central

    Tietz, Stefanie; Puthiyaveetil, Sujith; Enlow, Heather M.; Yarbrough, Robert; Wood, Magnus; Semchonok, Dmitry A.; Lowry, Troy; Li, Zhirong; Jahns, Peter; Boekema, Egbert J.; Lenhert, Steven; Niyogi, Krishna K.; Kirchhoff, Helmut

    2015-01-01

    The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion. PMID:25897076

  15. High-throughput production of prokaryotic membrane proteins.

    PubMed

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

    2005-01-01

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

  16. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  17. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  18. Compact Apparatus Grows Protein Crystals

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.; Delucas, Lawrence J.; Suddath, Fred L.; Snyder, Robert S.; Herren, Blair J.; Carter, Daniel C.; Yost, Vaughn H.

    1989-01-01

    Laboratory apparatus provides delicately balanced combination of materials and chemical conditions for growth of protein crystals. Apparatus and technique for growth based on hanging-drop method for crystallization of macromolecules. Includes pair of syringes with ganged plungers. One syringe contains protein solution; other contains precipitating-agent solution. Syringes intrude into cavity lined with porous reservoir material saturated with 1 mL or more of similar precipitating-agent solution. Prior to activation, ends of syringes plugged to prevent transport of water vapor among three solutions.

  19. 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

  20. Commercial Protein Crystal Growth: Protein Crystallization Facility (CPCG-H)

    NASA Astrophysics Data System (ADS)

    DeLucas, Lawrence J.

    2002-12-01

    Within the human body, there are thousands of different proteins that serve a variety of different functions, such as making it possible for red blood cells to carry oxygen in our bodies. Yet proteins can also be involved in diseases. Each protein has a particular chemical structure, which means it has a unique shape. It is this three-dimensional shape that allows each protein to do its job by interacting with chemicals or binding with other proteins. If researchers can determine the shape, or shapes, of a protein, they can learn how it works. This information can then be used by the pharmaceutical industry to develop new drugs or improve the way medications work. The NASA Commercial Space Center sponsoring this experiment - the Center for Biophysical Sciences and Engineering at the University of Alabama at Birmingham - has more than 60 industry and academic partners who grow protein crystals and use the information in drug design projects.

  1. 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.

  2. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

    The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

  3. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  4. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  5. The MORPHEUS protein crystallization screen.

    PubMed

    Gorrec, Fabrice

    2009-12-01

    A 96-condition initial screen for protein crystallization, called MORPHEUS, has been developed at the MRC Laboratory of Molecular Biology, Cambridge, England (MRC-LMB). The concept integrates several innovative approaches, such as chemically compatible mixes of potential ligands, new buffer systems and precipitant mixes that also act as cryoprotectants. Instead of gathering a set of crystallization conditions that have already been successful, a selection of molecules frequently observed in the Protein Data Bank (PDB) to co-crystallize with proteins has been made. These have been put together in mixes of similar chemical behaviour and structure, and combined with buffers and precipitant mixes that were also derived from PDB searches, to build the screen de novo. Observations made at the MRC-LMB and many practical aspects were also taken into account when formulating the screen. The resulting screen is easy to use, comprehensive yet small, and has already yielded a list of crystallization hits using both known and novel samples. As an indicator of success, the screen has now become one of the standard screens used routinely at the MRC-LMB when searching initial crystallization conditions for biological macromolecules.

  6. Automated protein crystal growth facility

    NASA Technical Reports Server (NTRS)

    Donald, Stacey

    1994-01-01

    A customer for the protein crystal growth facility fills the specially designed chamber with the correct solutions, fills the syringes with their quenching solutions, and submits the data needed for the proper growth of their crystal. To make sure that the chambers and syringes are filled correctly, a NASA representative may assist the customer. The data needed is the approximate growth time, the growth temperature, and the desired crystal size, but this data can be changed anytime from the ground, if needed. The chambers are gathered and placed into numbered slots in special drawers. Then, data is entered into a computer for each of the chambers. Technicians map out when each chamber's growth should be activated so that all of the chambers have enough time to grow. All of this data is up-linked to the space station when the previous growth session is over. Anti-vibrational containers need to be constructed for the high forces encountered during the lift off and the landing of the space shuttle, and though our team has not designed these containers, we do not feel that there is any reason why a suitable one could not be made. When the shuttle reaches the space station, an astronaut removes a drawer of quenched chambers from the growth facility and inserts a drawer of new chambers. All twelve of the drawers can be replaced in this fashion. The optical disks can also be removed this way. The old drawers are stored for the trip back to earth. Once inside the growth facility, a chamber is removed by the robot and placed in one of 144 active sites at a time previously picked by a technician. Growth begins when the chamber is inserted into an active site. Then, the sensing system starts to determine the size of the protein crystal. All during the crystal's growth, the customer can view the crystal and read all of the crystal's data, such as growth rate and crystal size. When the sensing system determines that the crystal has reached the predetermined size, the robot is

  7. Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

    NASA Technical Reports Server (NTRS)

    Feigelson, R. S. (Editor)

    1986-01-01

    Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

  8. Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

    NASA Technical Reports Server (NTRS)

    Feigelson, R. S. (Editor)

    1986-01-01

    Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

  9. The Crystal Structure of GXGD Membrane Protease FlaK

    SciTech Connect

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  10. The crystal structure of GXGD membrane protease FlaK

    SciTech Connect

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  11. 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.

  12. 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.

  13. Biopores/membrane proteins in synthetic polymer membranes.

    PubMed

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

    2017-04-01

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

  14. Slow cooling of protein crystals.

    PubMed

    Warkentin, Matthew; Thorne, Robert E

    2009-10-01

    Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s(-1) - 10(3)-10(4) times slower than in conventional flash cooling - while continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2 degrees . Most of the mosaicity increase occurs above approximately 200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3-6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.

  15. Microfluidic Approaches for Protein Crystal Structure Analysis.

    PubMed

    Maeki, Masatoshi; Yamaguchi, Hiroshi; Tokeshi, Manabu; Miyazaki, Masaya

    2016-01-01

    This review summarizes two microfluidic-based protein crystallization methods, protein crystallization behavior in the microfluidic devices, and their applications for X-ray crystal structure analysis. Microfluidic devices provide many advantages for protein crystallography; they require small sample volumes, provide high-throughput screening, and allow control of the protein crystallization. A droplet-based protein crystallization method is a useful technique for high-throughput screening and the formation of a single crystal without any complicated device fabrication process. Well-based microfluidic platforms also enable effective protein crystallization. This review also summarizes the protein crystal growth behavior in microfluidic devices as, is known from viewpoints of theoretical and experimental approaches. Finally, we introduce applications of microfluidic devices for on-chip crystal structure analysis.

  16. Automated high-throughput nanoliter-scale protein crystallization screening.

    PubMed

    Li, Fenglei; Robinson, Howard; Yeung, Edward S

    2005-12-01

    A highly efficient method is developed for automated high-throughput screening of nanoliter-scale protein crystallization. The system integrates liquid dispensing, crystallization and detection. The automated liquid dispensing system handles nanoliters of protein and various combinations of precipitants in parallel to access diverse regions of the phase diagram. A new detection scheme, native fluorescence, with complementary visible-light detection is employed for monitoring the progress of crystallization. This detection mode can distinguish protein crystals from inorganic crystals in a nondestructive manner. A gas-permeable membrane covering the microwells simplifies evaporation rate control and probes extended conditions in the phase diagram. The system was successfully demonstrated for the screening of lysozyme crystallization under 81 different conditions.

  17. 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

  18. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

    PubMed Central

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  19. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    PubMed

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-11-13

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

  20. Orientation and conformation of lipids in crystals of transmembrane proteins.

    PubMed

    Marsh, Derek; Páli, Tibor

    2013-03-01

    Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid-protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C-C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C-C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration.

  1. Studying how protein crystals form

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Watching molecules of the iron-storing protein apoferritin come together to form a nucleus reveals some interesting behavior. In this series of images, researchers observed clusters of four molecules at the corners of a diamond shape (top). As more molecules attach to the cluster, they arrange themselves into rods (second from top), and a raft-like configuration of molecules forms the critical nucleus (third from top), suggesting that crystal growth is much slower than it could be were the molecules arranged in a more compact formation. In the final image, a crystallite consisting of three layers containing approximately 60 to 70 molecules each is formed. Atomic force microscopy made visualizing the process of nucleation possible for the first time. The principal investigator is Peter Vekilov, of the University of Alabama in Huntsville. Vekilov's team at UAH studies protein solutions as they change phases from liquids to crystalline solids. They want to know if the molecules in the solution interact with one another, and if so, how, from the perspectives of thermodynamics and kinetics. They want to understand which forces -- electrical, electrostatic, hydrodynamic, or other kinds of forces -- are responsible for the interactions. They also study nucleation, the begirning stage of crystallization. This process is important to understand because it sets the stage for crystal growth in all kinds of solutions and liquid melts that are important in such diverse fields as agriculture, medicine, and the fabrication of metal components. Nucleation can determine the rate of crystal growth, the number of crystals that will be formed, and the quality and size of the crystals.

  2. Studying how protein crystals form

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Watching molecules of the iron-storing protein apoferritin come together to form a nucleus reveals some interesting behavior. In this series of images, researchers observed clusters of four molecules at the corners of a diamond shape (top). As more molecules attach to the cluster, they arrange themselves into rods (second from top), and a raft-like configuration of molecules forms the critical nucleus (third from top), suggesting that crystal growth is much slower than it could be were the molecules arranged in a more compact formation. In the final image, a crystallite consisting of three layers containing approximately 60 to 70 molecules each is formed. Atomic force microscopy made visualizing the process of nucleation possible for the first time. The principal investigator is Peter Vekilov, of the University of Alabama in Huntsville. Vekilov's team at UAH studies protein solutions as they change phases from liquids to crystalline solids. They want to know if the molecules in the solution interact with one another, and if so, how, from the perspectives of thermodynamics and kinetics. They want to understand which forces -- electrical, electrostatic, hydrodynamic, or other kinds of forces -- are responsible for the interactions. They also study nucleation, the begirning stage of crystallization. This process is important to understand because it sets the stage for crystal growth in all kinds of solutions and liquid melts that are important in such diverse fields as agriculture, medicine, and the fabrication of metal components. Nucleation can determine the rate of crystal growth, the number of crystals that will be formed, and the quality and size of the crystals.

  3. 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.

  4. Membrane shape modulates transmembrane protein distribution

    PubMed Central

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

    2014-01-01

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

  5. Approaches to automated protein crystal harvesting

    SciTech Connect

    Deller, Marc C. Rupp, Bernhard

    2014-01-28

    Approaches to automated and robot-assisted harvesting of protein crystals are critically reviewed. While no true turn-key solutions for automation of protein crystal harvesting are currently available, systems incorporating advanced robotics and micro-electromechanical systems represent exciting developments with the potential to revolutionize the way in which protein crystals are harvested.

  6. Tuning membrane protein mobility by confinement into nanodomains

    NASA Astrophysics Data System (ADS)

    Karner, Andreas; Nimmervoll, Benedikt; Plochberger, Birgit; Klotzsch, Enrico; Horner, Andreas; Knyazev, Denis G.; Kuttner, Roland; Winkler, Klemens; Winter, Lukas; Siligan, Christine; Ollinger, Nicole; Pohl, Peter; Preiner, Johannes

    2017-03-01

    High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

  7. 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

  8. Crystal structure of the integral membrane diacylglycerol kinase

    PubMed Central

    Li, Dianfan; Lyons, Joseph A.; Pye, Valerie E.; Vogeley, Lutz; Aragão, David; Kenyon, Colin P.; Shah, Syed T. A.; Doherty, Christine; Aherne, Margaret; Caffrey, Martin

    2013-01-01

    Diacylglycerol kinase (DgkA) catalyzes the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology1,3-7, folding8,9, assembly10-13, and stability1,14. Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT). These reveal a homo-trimeric enzyme with three transmembrane helices and an N-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site which is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are however at variance with a published solution NMR model2 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures. PMID:23676677

  9. Determination of Phase Diagrams for Soluble and Membrane Protein Systems

    SciTech Connect

    Talreja, S.; Perry, S; Guha, S; Zukoski, C; Kenis, P

    2010-01-01

    Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin.

  10. Determination of the Phase Diagram for Soluble and Membrane Proteins

    PubMed Central

    2010-01-01

    Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin. PMID:20235520

  11. Can Solution Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

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

    PubMed Central

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

    1970-01-01

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

  13. Membrane proteins: always an insoluble problem?

    PubMed Central

    Rawlings, Andrea E.

    2016-01-01

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

  14. Protein crystal growth in a microgravity environment

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1988-01-01

    Protein crystal growth is a major experimental problem and is the bottleneck in widespread applications of protein crystallography. Research efforts now being pursued and sponsored by NASA are making fundamental contributions to the understanding of the science of protein crystal growth. Microgravity environments offer the possibility of performing new types of experiments that may produce a better understanding of protein crystal growth processes and may permit growth environments that are more favorable for obtaining high quality protein crystals. A series of protein crystal growth experiments using the space shuttle was initiated. The first phase of these experiments was focused on the development of micro-methods for protein crystal growth by vapor diffusion techniques, using a space version of the hanging drop method. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth.

  15. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  16. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  17. 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.

  18. Structuring detergents for extracting and stabilizing functional membrane proteins.

    PubMed

    Matar-Merheb, Rima; Rhimi, Moez; Leydier, Antoine; Huché, Frédéric; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W; Falson, Pierre

    2011-03-31

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

  19. Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins

    PubMed Central

    Matar-Merheb, Rima; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W.; Falson, Pierre

    2011-01-01

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

  20. 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.

  1. 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.

  2. Protein-Induced Membrane Curvature Alters Local Membrane Tension

    PubMed Central

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

    2014-01-01

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

  3. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  4. Laser Irradiated Growth of Protein Crystal

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Takano, Kazufumi; Hosokawa, Youichiroh; Inoue, Tsuyoshi; Mori, Yusuke; Matsumura, Hiroyoshi; Yoshimura, Masashi; Tsunaka, Yasuo; Morikawa, Masaaki; Kanaya, Shigenori; Masuhara, Hiroshi; Kai, Yasushi; Sasaki, Takatomo

    2003-07-01

    We succeeded in the first ever generation of protein crystals by laser irradiation. We call this process Laser Irradiated Growth Technique (LIGHT). Effective crystallization was confirmed by applying an intense femtosecond laser. The crystallization period was dramatically shortened by LIGHT. In addition, protein crystals were obtained by LIGHT from normally uncrystallized conditions. These results indicate that intense femtosecond laser irradiation generates crystal nuclei; protein crystals can then be grown from the nuclei that act as seeds in a supersaturated solution. The nuclei formation is possible primarily due to nonlinear nucleation processes of an intense femtosecond laser with a peak intensity of over a gigawatt (GW).

  5. 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

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

    PubMed

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

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  7. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1991-01-01

    The objective of this research is to study the effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality. The application of graphoepitaxy (artificial epitaxy) to proteins is detailed. The development of a method for the control of nucleation is discussed. The factor affecting the morphology of isocitrate lyase crystals is presented.

  8. Integrated Protein-Crystal-Growing Apparatus

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.; Pusey, Marc L.

    1991-01-01

    Proposed apparatus for research on growth of protein crystals dispenses drops of protein and precipitating solutions, provides controlled environment for crystalization, and stores crystals. Intended for use in microgravity of outer space, concept of apparatus also useful in design of self-contained terrestrial experiments for remote and/or automatic execution.

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

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

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

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

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

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

  11. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

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

  12. DNA Nanotubes for NMR Structure Determination of Membrane Proteins

    PubMed Central

    Bellot, Gaëtan; McClintock, Mark A.; Chou, James J; Shih, William M.

    2013-01-01

    Structure determination of integral membrane proteins by solution NMR represents one of the most important challenges of structural biology. A Residual-Dipolar-Coupling-based refinement approach can be used to solve the structure of membrane proteins up to 40 kDa in size, however, a weak-alignment medium that is detergent-resistant is required. Previously, availability of media suitable for weak alignment of membrane proteins was severely limited. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400nm-long six-helix bundles each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, towards collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes via counter ions and small DNA binding molecules. This detergent-resistant liquid-crystal media offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility, and structural programmability. Production of sufficient nanotubes for 4–5 NMR experiments can be completed in one week by a single individual. PMID:23518667

  13. Measurements of Protein Crystal Face Growth Rates

    NASA Technical Reports Server (NTRS)

    Gorti, S.

    2014-01-01

    Protein crystal growth rates will be determined for several hyperthermophile proteins.; The growth rates will be assessed using available theoretical models, including kinetic roughening.; If/when kinetic roughening supersaturations are established, determinations of protein crystal quality over a range of supersaturations will also be assessed.; The results of our ground based effort may well address the existence of a correlation between fundamental growth mechanisms and protein crystal quality.

  14. Tetra Detector Analysis of Membrane Proteins

    PubMed Central

    Robbins, Rebecca A.; Stroud, Robert M.

    2014-01-01

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

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

    PubMed

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

    2008-05-01

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

  16. 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

  17. 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

  18. Tuning microbial hosts for membrane protein production

    PubMed Central

    2009-01-01

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

  19. Rapid protein crystallization by a micro osmotic screening system

    NASA Astrophysics Data System (ADS)

    Chan, Po-Hsiung; Su, Yu-Chuan

    2007-03-01

    This work presents a micro osmotic screening system that grows protein crystals in hours while consuming only micrograms of samples. Throughout the crystallization process, water can be driven in or out of a protein solution (across a semi-permeable membrane) to adjust its concentrations as desired. With the bi-directional and adjustable flow control realized by osmosis, each protein sample can be screened for crystallization conditions over a highly extended range. In the prototype demonstration, 6 × 8 screening arrays having an overall size of 20 × 24 × 2.5 mm3 were fabricated and characterized with crystallization experiments. In these experiments, crystallization conditions for four proteins, including lysozyme, catalase, thaumatin and xylanase, were identified within 2-6 h while consuming less than 20 µl of sample solution for each protein. Furthermore, it was also demonstrated that diffraction-quality crystals may be grown and harvested from the prototype system. As such, this osmotic system pioneers a new class of rapid screening schemes for high-throughput protein crystallization. A portion of this paper was presented at the 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Tokyo, Japan, November 2006.

  20. Crystal cataracts: Human genetic cataract caused by protein crystallization

    NASA Astrophysics Data System (ADS)

    Pande, Ajay; Pande, Jayanti; Asherie, Neer; Lomakin, Aleksey; Ogun, Olutayo; King, Jonathan; Benedek, George B.

    2001-05-01

    Several human genetic cataracts have been linked recently to point mutations in the D crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of D crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

  1. The X-ray structure of NccX from Cupriavidus metallidurans 31A illustrates potential dangers of detergent solubilization when generating and interpreting crystal structures of membrane proteins.

    PubMed

    Ziani, Widade; Maillard, Antoine P; Petit-Härtlein, Isabelle; Garnier, Norbert; Crouzy, Serge; Girard, Eric; Covès, Jacques

    2014-11-07

    The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 Å. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions.

  2. The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane Proteins

    PubMed Central

    Ziani, Widade; Maillard, Antoine P.; Petit-Härtlein, Isabelle; Garnier, Norbert; Crouzy, Serge; Girard, Eric; Covès, Jacques

    2014-01-01

    The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 Å. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions. PMID:25258316

  3. Protein-crystal growth experiment (planned)

    NASA Technical Reports Server (NTRS)

    Fujita, S.; Asano, K.; Hashitani, T.; Kitakohji, T.; Nemoto, H.; Kitamura, S.

    1988-01-01

    To evaluate the effectiveness of a microgravity environment on protein crystal growth, a system was developed using 5 cubic feet Get Away Special payload canister. In the experiment, protein (myoglobin) will be simultaneously crystallized from an aqueous solution in 16 crystallization units using three types of crystallization methods, i.e., batch, vapor diffusion, and free interface diffusion. Each unit has two compartments: one for the protein solution and the other for the ammonium sulfate solution. Compartments are separated by thick acrylic or thin stainless steel plates. Crystallization will be started by sliding out the plates, then will be periodically recorded up to 120 hours by a still camera. The temperature will be passively controlled by a phase transition thermal storage component and recorded in IC memory throughout the experiment. Microgravity environment can then be evaluated for protein crystal growth by comparing crystallization in space with that on Earth.

  4. Applications of the second virial coefficient: protein crystallization and solubility.

    PubMed

    Wilson, William W; Delucas, Lawrence J

    2014-05-01

    This article begins by highlighting some of the ground-based studies emanating from NASA's Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput.

  5. Applications of the second virial coefficient: protein crystallization and solubility

    PubMed Central

    Wilson, William W.; DeLucas, Lawrence J.

    2014-01-01

    This article begins by highlighting some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput. PMID:24817708

  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. [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. Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.

    PubMed

    Zhu, Ying; Zhu, Li-Na; Guo, Rui; Cui, Heng-Jun; Ye, Sheng; Fang, Qun

    2014-05-23

    Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35-96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4-8 nL. The protein consumption significantly reduces 50-500 fold compared with current crystallization stations.

  9. Nanoliter-Scale Protein Crystallization and Screening with a Microfluidic Droplet Robot

    PubMed Central

    Zhu, Ying; Zhu, Li-Na; Guo, Rui; Cui, Heng-Jun; Ye, Sheng; Fang, Qun

    2014-01-01

    Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35–96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4–8 nL. The protein consumption significantly reduces 50–500 fold compared with current crystallization stations. PMID:24854085

  10. 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.

  11. Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

    PubMed Central

    Gerdts, Cory J.; Stahl, Glenn L.; Napuli, Alberto; Staker, Bart; Abendroth, Jan; Edwards, Thomas E.; Myler, Peter; Van Voorhis, Wesley; Nollert, Peter; Stewart, Lance J.

    2010-01-01

    The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported. PMID:22477778

  12. Method for controlling protein crystallization

    NASA Technical Reports Server (NTRS)

    Noever, David A. (Inventor)

    1993-01-01

    A method and apparatus for controlling the crystallization of protein by solvent evaporation including placing a drop of protein solution between and in contact with a pair of parallel plates and driving one of the plates toward and away from the other plate in a controlled manner to adjust the spacing between the plates is presented. The drop of solution forms a liquid cylinder having a height dependent upon the plate spacing thereby effecting the surface area available for solvent evaporation. When the spacing is close, evaporation is slow. Evaporation is increased by increasing the spacing between the plates until the breaking point of the liquid cylinder. One plate is mounted upon a fixed post while the other plate is carried by a receptacle movable relative to the post and driven by a belt driven screw drive. The temperature and humidity of the drop of protein solution are controlled by sealing the drop within the receptacle and mounting a heater and dessicant within the receptacle.

  13. 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

  14. Gold nanoparticle capture within protein crystal scaffolds

    NASA Astrophysics Data System (ADS)

    Kowalski, Ann E.; Huber, Thaddaus R.; Ni, Thomas W.; Hartje, Luke F.; Appel, Karina L.; Yost, Jarad W.; Ackerson, Christopher J.; Snow, Christopher D.

    2016-06-01

    DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was

  15. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1989-01-01

    The mechanisms involved in protein crystallization and those parameters which influence the growth process and crystalline perfection were studied. The analysis of the flows around growing crystals is detailed. The preliminary study of the growth of isocitrate lyase and the crystal morphologies found are discussed. Preliminary results of controlled nucleation studies are presented.

  16. Compact Apparatus For Growth Of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Miller, Teresa Y.

    1991-01-01

    Compact apparatus proposed specifically for growth of protein crystals in microgravity also used in terrestrial laboratories to initiate and terminate growth at prescribed times automatically. Has few moving parts. Also contains no syringes difficult to clean, load, and unload and introduces contaminant silicon grease into crystallization solution. After growth of crystals terminated, specimens retrieved and transported simply.

  17. 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. Copyright © 2015. Published by Elsevier B.V.

  18. Improving the Quality of Protein Crystals Using Stirring Crystallization

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Matsumura, Hiroyoshi; Niino, Ai; Takano, Kazufumi; Kinoshita, Takayoshi; Warizaya, Masaichi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

    2004-04-01

    Recent reports state that a high magnetic field improves the crystal quality of bovine adenosine deaminase (ADA) with an inhibitor [Kinoshita et al.: Acta Cryst. D59 (2003) 1333]. In this paper, we examine the effect of stirring solution on ADA crystallization using a vapor-diffusion technique with rotary and figure-eight motion shakers. The probability of obtaining high-quality crystals is increased with stirring in a figure-eight pattern. Furthermore, rotary stirring greatly increased the probability of obtaining high-quality crystals, however, nucleation time was also increased. The crystal structure with the inhibitor was determined at a high resolution using a crystal obtained from a stirred solution. These results indicate that stirring with simple equipment is as useful as the high magnetic field technique for protein crystallization.

  19. 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.

  20. 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.

  1. The MORPHEUS II protein crystallization screen.

    PubMed

    Gorrec, Fabrice

    2015-07-01

    High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  2. Growth of shaped single crystals of proteins

    NASA Astrophysics Data System (ADS)

    Moreno, Abel; Rondón, Deyanira; García-Ruiz, Juan Ma.

    1996-09-01

    We present a procedure for obtaining protein single crystals that fill the capillary tubes in which they grow. The implementation was typical of the gel acupuncture method and the four different proteins are used as examples: lysozyme (HEW), thaumatin I, ferritin and insulin. Rod- and prismatic-shaped protein single crystals of these four proteins were grown inside capillary tubes of 0.2, 0.3, 0.5 mm in diameter and, for the case of lysozyme, up to 1.2 mm in diameter. The maximum length measured along the long axes of the rod crystals was 1.6 mm again for lysozyme crystals. It was observed that, once the capillary tube was filled, the crystal continues to grow by diffusion of the precipitating agent throughout the porous network formed by the protein crystal structure. We also discuss the possibility of growing these cylinders of crystalline proteins by the addition of protein solution to the mother liquor through the upper end of the glass capillary while the precipitating agent diffuses through the protein crystal itself. X-ray diffraction patterns confirm the single crystal character of the protein rods.

  3. Accelerated protein crystal growth by protein thin film template

    NASA Astrophysics Data System (ADS)

    Pechkova, Eugenia; Nicolini, Claudio

    2001-11-01

    A new method based on Langmuir-Blodgett (LB) technology is presented for the template stimulation of protein crystal growth. The new approach allows the acceleration of the hen egg white lysozyme (HEWL) crystal growth rate in comparison with such a classical vapour diffusion method as hanging drop. Protein thin films were coated on the cover slide of the common crystallization plates. Lysozyme crystal growth was observed on the LB thin films of HEWL.

  4. Applications of the second virial coefficient: protein crystallization and solubility

    SciTech Connect

    Wilson, William W.; DeLucas, Lawrence J.

    2014-04-30

    This article highlights some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program, and includes a more detailed discussion of the history and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. This article begins by highlighting some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput.

  5. Approaches to automated protein crystal harvesting

    PubMed Central

    Deller, Marc C.; Rupp, Bernhard

    2014-01-01

    The harvesting of protein crystals is almost always a necessary step in the determination of a protein structure using X-ray crystallographic techniques. However, protein crystals are usually fragile and susceptible to damage during the harvesting process. For this reason, protein crystal harvesting is the single step that remains entirely dependent on skilled human intervention. Automation has been implemented in the majority of other stages of the structure-determination pipeline, including cloning, expression, purification, crystallization and data collection. The gap in automation between crystallization and data collection results in a bottleneck in throughput and presents unfortunate opportunities for crystal damage. Several automated protein crystal harvesting systems have been developed, including systems utilizing microcapillaries, microtools, microgrippers, acoustic droplet ejection and optical traps. However, these systems have yet to be commonly deployed in the majority of crystallography laboratories owing to a variety of technical and cost-related issues. Automation of protein crystal harvesting remains essential for harnessing the full benefits of fourth-generation synchrotrons, free-electron lasers and microfocus beamlines. Furthermore, automation of protein crystal harvesting offers several benefits when compared with traditional manual approaches, including the ability to harvest microcrystals, improved flash-cooling procedures and increased throughput. PMID:24637746

  6. Which strategy for a protein crystallization project?

    NASA Technical Reports Server (NTRS)

    Kundrot, C. E.

    2004-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  7. Which Strategy for a Protein Crystallization Project?

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.

    2003-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  8. Advanced protein crystal growth programmatic sensitivity study

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The purpose of this study is to define the costs of various APCG (Advanced Protein Crystal Growth) program options and to determine the parameters which, if changed, impact the costs and goals of the programs and to what extent. This was accomplished by developing and evaluating several alternate programmatic scenarios for the microgravity Advanced Protein Crystal Growth program transitioning from the present shuttle activity to the man tended Space Station to the permanently manned Space Station. These scenarios include selected variations in such sensitivity parameters as development and operational costs, schedules, technology issues, and crystal growth methods. This final report provides information that will aid in planning the Advanced Protein Crystal Growth Program.

  9. Effect of Stirring Method on Protein Crystallization

    NASA Astrophysics Data System (ADS)

    Yaoi, Mari; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

    2004-10-01

    We previously proposed the use of solution stirring during the growth of protein crystals using the Micro-Stirring technique with a rotary shaker. In this paper, we report on the effects of a new type solution flow on the crystallization of hen egg-white lysozyme (HEWL) using a wave shaker. The time required for nucleation was reduced by wave stirring, but increased by rotary stirring. Nucleation was stimulated by wave stirring. This result indicates that protein crystal growth in a stirred solution is strongly dependent on the stirring method used and the solution flow. Therefore, optimized stirring conditions are essential for producing high-quality protein crystals.

  10. Which Strategy for a Protein Crystallization Project?

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.

    2003-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  11. Which strategy for a protein crystallization project?

    NASA Technical Reports Server (NTRS)

    Kundrot, C. E.

    2004-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  12. IFITM Proteins Restrict Viral Membrane Hemifusion

    PubMed Central

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

    2013-01-01

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

  13. Growth Of Oriented Crystals At Polymerized Membranes

    DOEpatents

    Charych, Deborah H. , Berman, Amir

    2000-01-25

    The present invention relates to methods and compositions for the growth and alignment of crystals at biopolymeric films. The methods and compositions of the present invention provide means to generate a variety of dense crystalline ceramic films, with totally aligned crystals, at low temperatures and pressures, suitable for use with polymer and plastic substrates.

  14. Crystallization of viruses and virus proteins

    NASA Astrophysics Data System (ADS)

    Sehnke, Paul C.; Harrington, Melissa; Hosur, M. V.; Li, Yunge; Usha, R.; Craig Tucker, R.; Bomu, Wu; Stauffacher, Cynthia V.; Johnson, John E.

    1988-07-01

    Methods for crystallizing six isometric plant and insect viruses are presented. Procedures developed for modifying, purifying and crystallizing coat protein subunits isolated from a virus forming asymmetric, spheroidal particles, stabilized almost exclusively by protein-RNA interactions, are also discussed. The tertiary and quaternary structures of small RNA viruses are compared.

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

    PubMed

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

    2006-10-07

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

  16. 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.

  17. 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.

  18. Membrane injury by pore-forming proteins.

    PubMed

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

    2009-08-01

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

  19. Cavity optomechanics with 2D photonic crystal membrane reflectors

    NASA Astrophysics Data System (ADS)

    Lingaraju, Navin B.; Shuai, Yichen; Lawall, John

    2016-03-01

    Membranes made from silicon nitride have significantly higher mechanical Q-factors under tensile stress than those made of other dielectric materials. This makes them ideal candidates for membrane reflectors that provide high finesse in Fabry-Perot cavities or membrane-in-the-middle optomechanical systems. Building on our previous work with one-dimensional gratings on suspended membranes, we patterned two-dimensional photonic crystal gratings on monolithic, suspended membranes made from silicon nitride. These high-Q membranes exhibited high reflectivity, upwards of 99%, over several nanometers in the telecom band. To probe their optical response in a cavity environment, we used these membrane reflectors as the moving mirror in a Fabry-Perot cavity. We were able to realize cavities with a finesse of over 4,500.

  20. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

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

    2015-01-01

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

  1. Protein crystal growth (5-IML-1)

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1992-01-01

    Proteins (enzymes, hormones, immunoglobulins) account for 50 pct. or more of the dry weight of most living systems. A detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting projects have terminated at the crystal growth stage. In principle, there are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor is the elimination of density driven convective flow. Other factors that can be controlled in the absence of gravity is the sedimentation of growing crystals in a gravitational field, and the potential advantage of doing containerless crystal growth. As a result of these theories and facts, one can readily understand why the microgravity environment of an Earth orbiting vehicle seems to offer unique opportunities for the protein crystallographer. This perception has led to the establishment of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project. The results of experiments already performed during STS missions have in many cases resulted in large protein crystals which are structurally correct. Thus, the near term objective of the PCG/ME project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  2. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1990-01-01

    The effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality was studied. The proper design of the flight hardware and experimental protocols are highly dependent on understanding the factors which influence the nucleation and growth of crystals of biological macromolecules. Thus, those factors are investigated and the body of knowledge which has been built up for small molecule crystallization. These data also provide a basis of comparison for the results obtained from low-g experiments. The flows around growing crystals are detailed. The preliminary study of the growth of isocitrate lyase, the crystal morphologies found and the preliminary x ray results are discussed. The design of two apparatus for protein crystal growth by temperature control are presented along with preliminary results.

  3. Crystal packing effects on protein loops.

    PubMed

    Rapp, Chaya S; Pollack, Rena M

    2005-07-01

    The effects of crystal packing on protein loop structures are examined by (1) a comparison of loops in proteins that have been crystallized in alternate packing arrangements, and (2) theoretical prediction of loops both with and without the inclusion of the crystal environment. Results show that in a minority of cases, loop geometries are dependent on crystal packing effects. Explicit representation of the crystal environment in a loop prediction algorithm can be used to model these effects and to reconstruct the structures, and relative energies, of a loop in alternative packing environments. By comparing prediction results with and without the inclusion of the crystal environment, the loop prediction algorithm can further be used to identify cases in which a crystal structure does not represent the most stable state of a loop in solution. We anticipate that this capability has implications for structural biology.

  4. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

    PubMed

    Bratanov, Dmitry; Balandin, Taras; Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.

  5. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

    PubMed Central

    Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. PMID:26046789

  6. The MORPHEUS II protein crystallization screen

    SciTech Connect

    Gorrec, Fabrice

    2015-06-27

    MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  7. 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.

  8. 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

  9. Polyene antibiotic that inhibits membrane transport proteins.

    PubMed

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

    2012-07-10

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

  10. Using Microfluidics to Decouple Nucleation and Growth of Protein Crystals.

    PubMed

    Shim, Jung-Uk; Cristobal, Galder; Link, Darren R; Thorsen, Todd; Fraden, Seth

    2007-01-01

    A high throughput, low volume microfluidic device has been designed to decouple the physical processes of protein crystal nucleation and growth. This device, called the Phase Chip, is constructed out of poly(dimethylsiloxane) (PDMS) elastomer. One of the Phase Chip's innovations is to exploit surface tension forces to guide each drop to a storage chamber. We demonstrate that nanoliter water-in-oil drops of protein solutions can be rapidly stored in individual wells thereby allowing the screening of 1000 conditions while consuming a total of only 10 mug protein on a 20 cm(2) chip. Another significant advance over current microfluidic devices is that each well is in contact with a reservoir via a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or protein. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current methods, such as the free interface diffusion or sitting drop methods, which are irreversible. The Phase Chip operates by first optimizing conditions for nucleation by using dialysis to supersaturate the protein solution, which leads to nucleation of many small crystals. Next, conditions are optimized for crystal growth by using dialysis to reduce the protein and precipitant concentrations, which leads small crystals to dissolve while simultaneously causing only the largest ones to grow, ultimately resulting in the transformation of many small, unusable crystals into a few large ones.

  11. Solid-state NMR and Membrane Proteins

    PubMed Central

    Opella, Stanley J.

    2015-01-01

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

  12. Solid-state NMR and membrane proteins

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2015-04-01

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

  13. The Protein Crystallization Facility STS-95

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The Protein Crystallization Facility will be used to grow crystals of human insulin. Insulin is the primary treatment for diabetes, the fourth leading cause of death by disease. Research on STS-95 is aimed at producing crystals of even higher quality, which when combined with new analysis techniques will permit a better understanding of the interaction between insulin and its receptor. This has the potential to aid in the development of a new commercially available insulin product with unique time release properties that could reduce fluctuations in a patient's blood sugar level. The Protein Crystallization Facility supports large-scale commercial investigations.

  14. The Protein Crystallization Facility STS-95

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The Protein Crystallization Facility will be used to grow crystals of human insulin. Insulin is the primary treatment for diabetes, the fourth leading cause of death by disease. Research on STS-95 is aimed at producing crystals of even higher quality, which when combined with new analysis techniques will permit a better understanding of the interaction between insulin and its receptor. This has the potential to aid in the development of a new commercially available insulin product with unique time release properties that could reduce fluctuations in a patient's blood sugar level. The Protein Crystallization Facility supports large-scale commercial investigations.

  15. Electrostatic Stabilization Of Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.

    1991-01-01

    Proposed technique produces large crystals in compact, economical apparatus. Report presents concept for supporting protein crystals during growth in microgravity. Yields crystals larger and more-nearly perfect than those grown on Earth. Combines best features of sandwich-drop and electrostatic-levitation methods of support. Drop of protein solution inserted between pair of glass or plastic plates, as in sandwich-drop-support method. Electrostatically charged ring confines drop laterally and shapes it, as in electrostatic technique. Apparatus also made to accommodate several drops simultaneously between same pair of supporting plates. Drops can be inserted and crystals removed through ducts in plates.

  16. Predictive control of crystal size distribution in protein crystallization.

    PubMed

    Shi, Dan; Mhaskar, Prashant; El-Farra, Nael H; Christofides, Panagiotis D

    2005-07-01

    This work focuses on the modelling, simulation and control of a batch protein crystallization process that is used to produce the crystals of tetragonal hen egg-white (HEW) lysozyme. First, a model is presented that describes the formation of protein crystals via nucleation and growth. Existing experimental data are used to develop empirical models of the nucleation and growth mechanisms of the tetragonal HEW lysozyme crystal. The developed growth and nucleation rate expressions are used within a population balance model to simulate the batch crystallization process. Then, model reduction techniques are used to derive a reduced-order moments model for the purpose of controller design. Online measurements of the solute concentration and reactor temperature are assumed to be available, and a Luenberger-type observer is used to estimate the moments of the crystal size distribution based on the available measurements. A predictive controller, which uses the available state estimates, is designed to achieve the objective of maximizing the volume-averaged crystal size while respecting constraints on the manipulated input variables (which reflect physical limitations of control actuators) and on the process state variables (which reflect performance considerations). Simulation results demonstrate that the proposed predictive controller is able to increase the volume-averaged crystal size by 30% and 8.5% compared to constant temperature control (CTC) and constant supersaturation control (CSC) strategies, respectively, while reducing the number of fine crystals produced. Furthermore, a comparison of the crystal size distributions (CSDs) indicates that the product achieved by the proposed predictive control strategy has larger total volume and lower polydispersity compared to the CTC and CSC strategies. Finally, the robustness of the proposed method (achieved due to the presence of feedback) with respect to plant-model mismatch is demonstrated. The proposed method is

  17. 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

  18. Protein Crystallization Using Room Temperature Ionic Fluids

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Paley, Mark Steve; Turner, Megan B.; Rogers, Robin D.

    2006-01-01

    The ionic liquids (ILs) 1-butyl-3-methylimidizolium chloride (C4mim-C1), 1-butyl-3- methylimidizolium diethyleneglycol monomethylethersulfate ([C4mim]DEMGS), and 1-butyl-1 -methylpyrollidinium dihydrogenphosphate ([p1,4]dhp) were tested for their effects on the crystallization of the proteins canavalin, beta-lactoglobulin B, xylanase, and glucose isomerase, using a standard high throughput screen. The crystallization experiments were set up with the ILs added to the protein solutions at 0.2 and 0.4 M final concentrations. Crystallization droplets were set up at three proteixprecipitant ratios (1:1, 2:1, and 4:l), which served to progressively dilute the effects of the screen components while increasing the equilibrium protein and IL concentrations. Crystals were obtained for all four proteins at a number of conditions where they were not obtained from the IL-free control experiment. Over half of the protein-IL combinations tested had more successful outcomes than negative, where the IL-free crystallization was better than the corresponding IL-containing outcome, relative to the control. One of the most common causes of a negative outcome was solubilization of the protein by the IL, resulting in a clear drop. In one instance, we were able to use the IL-induced solubilizing to obtain beta-lactoglobulin B crystals from conditions that gave precipitated protein in the absence of IL. The results suggest that it may be feasible to develop ILs specifically for the task of macromolecule crystallization.

  19. Protein Crystallization Using Room Temperature Ionic Fluids

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Paley, Mark Steve; Turner, Megan B.; Rogers, Robin D.

    2006-01-01

    The ionic liquids (ILs) 1-butyl-3-methylimidizolium chloride (C4mim-C1), 1-butyl-3- methylimidizolium diethyleneglycol monomethylethersulfate ([C4mim]DEMGS), and 1-butyl-1 -methylpyrollidinium dihydrogenphosphate ([p1,4]dhp) were tested for their effects on the crystallization of the proteins canavalin, beta-lactoglobulin B, xylanase, and glucose isomerase, using a standard high throughput screen. The crystallization experiments were set up with the ILs added to the protein solutions at 0.2 and 0.4 M final concentrations. Crystallization droplets were set up at three proteixprecipitant ratios (1:1, 2:1, and 4:l), which served to progressively dilute the effects of the screen components while increasing the equilibrium protein and IL concentrations. Crystals were obtained for all four proteins at a number of conditions where they were not obtained from the IL-free control experiment. Over half of the protein-IL combinations tested had more successful outcomes than negative, where the IL-free crystallization was better than the corresponding IL-containing outcome, relative to the control. One of the most common causes of a negative outcome was solubilization of the protein by the IL, resulting in a clear drop. In one instance, we were able to use the IL-induced solubilizing to obtain beta-lactoglobulin B crystals from conditions that gave precipitated protein in the absence of IL. The results suggest that it may be feasible to develop ILs specifically for the task of macromolecule crystallization.

  20. 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.

  1. Protein profiles of hatchery egg shell membrane

    USDA-ARS?s Scientific Manuscript database

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

  2. Detergents in Membrane Protein Purification and Crystallisation.

    PubMed

    Anandan, Anandhi; Vrielink, Alice

    2016-01-01

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

  3. Solid-state NMR structures of integral membrane proteins.

    PubMed

    Patching, Simon G

    2015-01-01

    Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.

  4. Renaturing Membrane Proteins in the Lipid Cubic Phase, a Nanoporous Membrane Mimetic

    PubMed Central

    Li, Dianfan; Caffrey, Martin

    2014-01-01

    Membrane proteins play vital roles in the life of the cell and are important therapeutic targets. Producing them in large quantities, pure and fully functional is a major challenge. Many promising projects end when intractable aggregates or precipitates form. Here we show how such unfolded aggregates can be solubilized and the solution mixed with lipid to spontaneously self-assemble a bicontinuous cubic mesophase into the bilayer of which the protein, in a confined, chaperonin-like environment, reconstitutes with 100% efficiency. The test protein, diacylglycerol kinase, reconstituted in the bilayer of the mesophase, was then crystallized in situ by the in meso or lipid cubic phase method providing an X-ray structure to a resolution of 2.55 Å. This highly efficient, inexpensive, simple and rapid approach should find application wherever properly folded, membrane reconstituted and functional proteins are required where the starting material is a denatured aggregate. PMID:25055873

  5. Polymer single crystal membrane from liquid/liquid interface

    NASA Astrophysics Data System (ADS)

    Wang, Wenda; Li, Christopher; Soft Matter Research Group-Drexel University Team

    2013-03-01

    Vesicles, mimicking the structure of cell membrane at the molecular scale, are small membrane-enclosed sacks that can store or transport substances. The weak mechanical properties and the nature of environment-sensitivity of the current available vesicles: liposomes, polymersomes, colloidsomes limit their applications as an excellent candidate for targeting delivery of drugs/genes in biomedical engineering and treatment. Recently, we developed an emulsion-based method to grow curved polymer single crystals. Varying the polymer concentration and/or the emulsification conditions (such as surfactant concentration, water-oil volume ratio), curved crystals with different sizes and different openness could be obtained. This growing process was attributed to polymer crystal growth along the liquid/liquid interface. In addition, the liquid/liquid interfacial crystal growth is promising for synthesis of enclosed hollow sphere.

  6. 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

  7. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

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

    2014-01-01

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

  8. Phonon dispersion in hypersonic two-dimensional phononic crystal membranes

    NASA Astrophysics Data System (ADS)

    Graczykowski, B.; Sledzinska, M.; Alzina, F.; Gomis-Bresco, J.; Reparaz, J. S.; Wagner, M. R.; Sotomayor Torres, C. M.

    2015-02-01

    We investigate experimentally and theoretically the acoustic phonon propagation in two-dimensional phononic crystal membranes. Solid-air and solid-solid phononic crystals were made of square lattices of holes and Au pillars in and on 250 nm thick single crystalline Si membrane, respectively. The hypersonic phonon dispersion was investigated using Brillouin light scattering. Volume reduction (holes) or mass loading (pillars) accompanied with second-order periodicity and local resonances are shown to significantly modify the propagation of thermally activated GHz phonons. We use numerical modeling based on the finite element method to analyze the experimental results and determine polarization, symmetry, or three-dimensional localization of observed modes.

  9. Protein profiles of hatchery egg shell membrane.

    PubMed

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

    2016-01-01

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

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

    PubMed

    Zeev-Ben-Mordehai, Tzviya; Weberruß, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G; Antonin, Wolfram; Mettenleiter, Thomas C; Grünewald, Kay

    2015-12-29

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

  11. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

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

    2008-09-23

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

  12. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

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

    2008-01-01

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

  13. Thermal crystallization mechanism of silk fibroin protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao

    In this thesis, the thermal crystallization mechanism of silk fibroin protein from Bombyx mori silkworm, was treated as a model for the general study of protein based materials, combining theories from both biophysics and polymer physics fields. A systematic and scientific path way to model the dynamic beta-sheet crystallization process of silk fibroin protein was presented in the following sequence: (1) The crystallinity, fractions of secondary structures, and phase compositions in silk fibroin proteins at any transition stage were determined. Two experimental methods, Fourier transform infrared spectroscopy (FTIR) with Fourier self-deconvolution, and specific reversing heat capacity, were used together for the first time for modeling the static structures and phases in the silk fibroin proteins. The protein secondary structure fractions during the crystallization were quantitatively determined. The possibility of existence of a "rigid amorphous phase" in silk protein was also discussed. (2) The function of bound water during the crystallization process of silk fibroin was studied using heat capacity, and used to build a silk-water dynamic crystallization model. The fundamental concepts and thermal properties of silk fibroin with/without bound water were discussed. Results show that intermolecular bound water molecules, acting as a plasticizer, will cause silk to display a water-induced glass transition around 80°C. During heating, water is lost, and the change of the microenvironment in the silk fibroin chains induces a mesophase prior to thermal crystallization. Real time FTIR during heating and isothermal holding above Tg show the tyrosine side chain changes only during the former process, while beta sheet crystallization occurs only during the latter process. Analogy is made between the crystallization of synthetic polymers according to the four-state scheme of Strobl, and the crystallization process of silk fibroin, which includes an intermediate precursor

  14. Toward structure determination using membrane-protein nanocrystals and microcrystals

    PubMed Central

    Hunter, Mark S.; Fromme, Petra

    2012-01-01

    Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (~ 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-Å wavelength, Bragg peaks were recorded to 8.5-Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [Chapman H.N., et al. Femtosecond X-ray protein nanocrystallography, Nature 470 (2011) 73–81]. Many additional techniques still need to be developed to explore the

  15. 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.

  16. 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

  17. The Nucleation and Growth of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc

    2004-01-01

    Obtaining crystals of suitable size and high quality continues to be a major bottleneck in macromolecular crystallography. Currently, structural genomics efforts are achieving on average about a 10% success rate in going from purified protein to a deposited crystal structure. Growth of crystals in microgravity was proposed as a means of overcoming size and quality problems, which subsequently led to a major NASA effort in microgravity crystal growth, with the agency also funding research into understanding the process. Studies of the macromolecule crystal nucleation and growth process were carried out in a number of labs in an effort to understand what affected the resultant crystal quality on Earth, and how microgravity improved the process. Based upon experimental evidence, as well as simple starting assumptions, we have proposed that crystal nucleation occurs by a series of discrete self assembly steps, which 'set' the underlying crystal symmetry. This talk will review the model developed, and its origins, in our laboratory for how crystals nucleate and grow, and will then present, along with preliminary data, how we propose to use this model to improve the success rate for obtaining crystals from a given protein.

  18. The Nucleation and Growth of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc

    2004-01-01

    Obtaining crystals of suitable size and high quality continues to be a major bottleneck in macromolecular crystallography. Currently, structural genomics efforts are achieving on average about a 10% success rate in going from purified protein to a deposited crystal structure. Growth of crystals in microgravity was proposed as a means of overcoming size and quality problems, which subsequently led to a major NASA effort in microgravity crystal growth, with the agency also funding research into understanding the process. Studies of the macromolecule crystal nucleation and growth process were carried out in a number of labs in an effort to understand what affected the resultant crystal quality on Earth, and how microgravity improved the process. Based upon experimental evidence, as well as simple starting assumptions, we have proposed that crystal nucleation occurs by a series of discrete self assembly steps, which 'set' the underlying crystal symmetry. This talk will review the model developed, and its origins, in our laboratory for how crystals nucleate and grow, and will then present, along with preliminary data, how we propose to use this model to improve the success rate for obtaining crystals from a given protein.

  19. Protein quality control at the plasma membrane

    PubMed Central

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

    2011-01-01

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

  20. Protein quality control at the plasma membrane.

    PubMed

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

    2011-08-01

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

  1. 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.

  2. (PCG) Protein Crystal Growth Porcine Elastase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Porcine Elastase. This enzyme is associated with the degradation of lung tissue in people suffering from emphysema. It is useful in studying causes of this disease. Principal Investigator on STS-26 was Charles Bugg.

  3. Frequency control of photonic crystal membrane resonators by monolayer deposition

    NASA Astrophysics Data System (ADS)

    Strauf, S.; Rakher, M. T.; Carmeli, I.; Hennessy, K.; Meier, C.; Badolato, A.; DeDood, M. J. A.; Petroff, P. M.; Hu, E. L.; Gwinn, E. G.; Bouwmeester, D.

    2006-01-01

    We study the response of GaAs photonic crystal membrane resonators to thin-film deposition. Slow spectral shifts of the cavity mode of several nanometers are observed at low temperatures, caused by cryo-gettering of background molecules. Heating the membrane resets the drift and shielding will prevent drift altogether. In order to explore the drift as a tool to detect surface layers, or to intentionally shift the cavity resonance frequency, we studied the effect of self-assembled monolayers of polypeptide molecules attached to the membranes. The 2-nm-thick monolayers lead to a discrete step in the resonance frequency and partially passivate the surface.

  4. Conformation of Membrane Proteins: Bacteriorhodopsin

    DTIC Science & Technology

    1991-12-09

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

  5. Can Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

  6. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  7. Optimization of buffer solutions for protein crystallization.

    PubMed

    Gosavi, Rajendrakumar A; Mueser, Timothy C; Schall, Constance A

    2008-05-01

    Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.

  8. Trace fluorescent labeling for protein crystallization

    PubMed Central

    Pusey, Marc; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-01-01

    Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent. PMID:26144224

  9. 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

  10. 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.

  11. 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.

  12. Convection effects in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Roberts, Glyn O.

    1988-01-01

    Protein crystals for X-ray diffraction study are usually grown resting on the bottom of a hanging drop of a saturated protein solution, with slow evaporation to the air in a small enclosed cell. The evaporation rate is controlled by hanging the drop above a reservoir of water, with its saturation vapor pressure decreased by a low concentration of a passive solute. The drop has a lower solute concentration, and its volume shrinks by evaporation until the molecular concentrations match. Protein crystals can also be grown from a seed crystal suspended or supported in the interior of a supersaturated solution. The main analysis of this report concerns this case because it is less complicated than hanging-drop growth. Convection effects have been suggested as the reason for the apparent cessation of growth at a certain rather small crystal size. It seeems that as the crystal grows, the number of dislocations increases to a point where further growth is hindered. Growth in the microgravity environment of an orbiting space vehicle has been proposed as a method for obtaining larger crystals. Experimental observations of convection effects during the growth of protein crystals have been reported.

  13. 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.

  14. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1993-01-01

    This Final Technical Report for NASA Grant NAG8-774 covers the period from April 27, 1989 through December 31, 1992. It covers five main topics: fluid flow studies, the influence of growth conditions on the morphology of isocitrate lyase crystals, control of nucleation, the growth of lysozyme by the temperature gradient method and graphoepitaxy of protein crystals. The section on fluid flow discusses the limits of detectability in the Schlieren imaging of fluid flows around protein crystals. The isocitrate lyase study compares crystals grown terrestrially under a variety of conditions with those grown in space. The controlling factor governing the morphology of the crystals is the supersaturation. The lack of flow in the interface between the drop and the atmosphere in microgravity causes protein precipitation in the boundary layer and a lowering of the supersaturation in the drop. This lowered supersaturation leads to improved crystal morphology. Preliminary experiments with lysozyme indicated that localized temperature gradients could be used to nucleate crystals in a controlled manner. An apparatus (thermonucleator) was designed to study the controlled nucleation of protein crystals. This apparatus has been used to nucleate crystals of materials with both normal (ice-water, Rochelle salt and lysozyme) and retrograde (horse serum albumin and alpha chymotrypsinogen A) solubility. These studies have lead to the design of an new apparatus that small and more compatible with use in microgravity. Lysozyme crystals were grown by transporting nutrient from a source (lysozyme powder) to the crystal in a temperature gradient. The influence of path length and cross section on the growth rate was demonstrated. This technique can be combined with the thermonucleator to control both nucleation and growth. Graphoepitaxy utilizes a patterned substrate to orient growing crystals. In this study, silicon substrates with 10 micron grooves were used to grow crystals of catalase

  15. Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Lee, Sin-Doo

    2015-10-01

    Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.

  16. Protein Crystallization: Specific Phenomena and General Insights on Crystallization Kinetics

    NASA Technical Reports Server (NTRS)

    Rosenberger, F.

    1998-01-01

    Experimental and simulation studies of the nucleation and growth kinetics of proteins have revealed phenomena that are specific for macromolecular crystallization, and others that provide a more detailed understanding of solution crystallization in general. The more specific phenomena, which include metastable liquid-liquid phase separations and gelation prior to solid nucleation, are due to the small ratio of the intermolecular interaction-range to the size of molecules involved. The apparently more generally applicable mechanisms include the cascade-like formation of macrosteps, as an intrinsic morphological instability that roots in the coupled bulk transport and nonlinear interface kinetics in systems with mixed growth rate control. Analyses of this nonlinear response provide (a) criteria for the choice of bulk transport conditions to minimize structural defect formation, and (b) indications that the "slow" protein crystallization kinetics stems from the mutual retardation of growth steps.

  17. Protein Crystallization: Specific Phenomena and General Insights on Crystallization Kinetics

    NASA Technical Reports Server (NTRS)

    Rosenberger, F.

    1998-01-01

    Experimental and simulation studies of the nucleation and growth kinetics of proteins have revealed phenomena that are specific for macromolecular crystallization, and others that provide a more detailed understanding of solution crystallization in general. The more specific phenomena, which include metastable liquid-liquid phase separations and gelation prior to solid nucleation, are due to the small ratio of the intermolecular interaction-range to the size of molecules involved. The apparently more generally applicable mechanisms include the cascade-like formation of macrosteps, as an intrinsic morphological instability that roots in the coupled bulk transport and nonlinear interface kinetics in systems with mixed growth rate control. Analyses of this nonlinear response provide (a) criteria for the choice of bulk transport conditions to minimize structural defect formation, and (b) indications that the "slow" protein crystallization kinetics stems from the mutual retardation of growth steps.

  18. Femtosecond crystallography of membrane proteins in the lipidic cubic phase

    PubMed Central

    Liu, Wei; Wacker, Daniel; Wang, Chong; Abola, Enrique; Cherezov, Vadim

    2014-01-01

    Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins. PMID:24914147

  19. 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.

  20. Trace fluorescent labeling for protein crystallization

    SciTech Connect

    Pusey, Marc Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-06-27

    The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.

  1. 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.

  2. 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.

  3. 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.

  4. Present and future of membrane protein structure determination by electron crystallography

    PubMed Central

    Ubarretxena-Belandia, Iban; Stokes, David L.

    2011-01-01

    Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

  5. Protein Crystal Growth Dynamics and Impurity Incorporation

    NASA Technical Reports Server (NTRS)

    Chernov, Alex A.; Thomas, Bill

    2000-01-01

    The general concepts and theories of crystal growth are proven to work for biomolecular crystallization. This allowed us to extract basic parameters controlling growth kinetics - free surface energy, alpha, and kinetic coefficient, beta, for steps. Surface energy per molecular site in thermal units, alpha(omega)(sup 2/3)/kT approx. = 1, is close to the one for inorganic crystals in solution (omega is the specific molecular volume, T is the temperature). Entropic restrictions on incorporation of biomolecules into the lattice reduce the incorporation rate, beta, by a factor of 10(exp 2) - 10(exp 3) relative to inorganic crystals. A dehydration barrier of approx. 18kcal/mol may explain approx. 10(exp -6) times difference between frequencies of adding a molecule to the lattice and Brownian attempts to do so. The latter was obtained from AFM measurements of step and kink growth rates on orthorhombic lysozyme. Protein and many inorganic crystals typically do not belong to the Kossel type, thus requiring a theory to account for inequivalent molecular positions within its unit cell. Orthorhombic lysozyme will serve as an example of how to develop such a theory. Factors deteriorating crystal quality - stress and strain, mosaicity, molecular disorder - will be reviewed with emphasis on impurities. Dimers in ferritin and lysozyme and acetylated lysozyme, are microheterogeneous i.e. nearly isomorphic impurities that are shown to be preferentially trapped by tetragonal lysozyme and ferritin crystals, respectively. The distribution coefficient, K defined as a ratio of the (impurity/protein) ratios in crystal and in solution is a measure of trapping. For acetylated lysoyzme, K = 2.15 or, 3.42 for differently acetylated forms, is independent of both the impurity and the crystallizing protein concentration. The reason is that impurity flux to the surface is constant while the growth rate rises with supersaturation. About 3 times lower dimer concentration in space grown ferritin and

  6. Protein Crystal Growth Dynamics and Impurity Incorporation

    NASA Technical Reports Server (NTRS)

    Chernov, Alex A.; Thomas, Bill

    2000-01-01

    The general concepts and theories of crystal growth are proven to work for biomolecular crystallization. This allowed us to extract basic parameters controlling growth kinetics - free surface energy, alpha, and kinetic coefficient, beta, for steps. Surface energy per molecular site in thermal units, alpha(omega)(sup 2/3)/kT approx. = 1, is close to the one for inorganic crystals in solution (omega is the specific molecular volume, T is the temperature). Entropic restrictions on incorporation of biomolecules into the lattice reduce the incorporation rate, beta, by a factor of 10(exp 2) - 10(exp 3) relative to inorganic crystals. A dehydration barrier of approx. 18kcal/mol may explain approx. 10(exp -6) times difference between frequencies of adding a molecule to the lattice and Brownian attempts to do so. The latter was obtained from AFM measurements of step and kink growth rates on orthorhombic lysozyme. Protein and many inorganic crystals typically do not belong to the Kossel type, thus requiring a theory to account for inequivalent molecular positions within its unit cell. Orthorhombic lysozyme will serve as an example of how to develop such a theory. Factors deteriorating crystal quality - stress and strain, mosaicity, molecular disorder - will be reviewed with emphasis on impurities. Dimers in ferritin and lysozyme and acetylated lysozyme, are microheterogeneous i.e. nearly isomorphic impurities that are shown to be preferentially trapped by tetragonal lysozyme and ferritin crystals, respectively. The distribution coefficient, K defined as a ratio of the (impurity/protein) ratios in crystal and in solution is a measure of trapping. For acetylated lysoyzme, K = 2.15 or, 3.42 for differently acetylated forms, is independent of both the impurity and the crystallizing protein concentration. The reason is that impurity flux to the surface is constant while the growth rate rises with supersaturation. About 3 times lower dimer concentration in space grown ferritin and

  7. Crystal structure of a defective folding protein

    PubMed Central

    Saul, Frederick A.; Mourez, Michaël; Vulliez-le Normand, Brigitte; Sassoon, Nathalie; Bentley, Graham A.; Betton, Jean-Michel

    2003-01-01

    Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32→Asp and Ile 33→Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 Å resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first α-helix to the first β-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system. PMID:12592028

  8. Crystal structure of a defective folding protein.

    PubMed

    Saul, Frederick A; Mourez, Michaël; Vulliez-Le Normand, Brigitte; Sassoon, Nathalie; Bentley, Graham A; Betton, Jean-Michel

    2003-03-01

    Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.

  9. 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

  10. 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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. 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.

  12. Transmembrane protein sorting driven by membrane curvature.

    PubMed

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

    2015-11-02

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

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

    PubMed Central

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

    2010-01-01

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

  14. Membrane-targeting liquid crystal nanoparticles (LCNPs) for drug delivery

    NASA Astrophysics Data System (ADS)

    Nag, Okhil K.; Naciri, Jawad; Spillmann, Christopher M.; Delehanty, James B.

    2016-03-01

    In addition to maintaining the structural integrity of the cell, the plasma membrane regulates multiple important cellular processes, such as endocytosis and trafficking, apoptotic pathways and drug transport. The modulation or tracking of such cellular processes by means of controlled delivery of drugs or imaging agents via nanoscale delivery systems is very attractive. Nanoparticle-mediated delivery systems that mediate long-term residence (e.g., days) and controlled release of the cargoes in the plasma membrane while simultaneously not interfering with regular cellular physiology would be ideal for this purpose. Our laboratory has developed a plasma membrane-targeted liquid crystal nanoparticle (LCNP) formulation that can be loaded with dyes or drugs which can be slowly released from the particle over time. Here we highlight the utility of these nanopreparations for membrane delivery and imaging.

  15. Super-thin single crystal diamond membrane radiation detectors

    SciTech Connect

    Pomorski, Michal; Caylar, Benoit; Bergonzo, Philippe

    2013-09-09

    We propose to use the non-electronic grade (nitrogen content 5 ppb < [N] < 5 ppm) single crystal (sc) chemical vapour deposited (CVD) diamond as a thin-membrane radiation detector. Using deep Ar/O{sub 2} plasma etching it is possible to produce self-supported few micrometres thick scCVD membranes of a size approaching 7 mm × 7 mm, with a very good surface quality. After metallization and contacting, electrical properties of diamond membrane detectors were probed with 5.486 MeV α-particles as an ionization source. Despite nitrogen impurity, scCVD membrane detectors exhibit stable operation, charge collection efficiency close to 100%, with homogenous response, and extraordinary dielectric strength up to 30 V/μm.

  16. Disorder in flash cooled protein crystals

    NASA Astrophysics Data System (ADS)

    Kriminski, Sergey Alekseevich

    Heat transfer from protein crystals and temperature distribution during flash cooling were analyzed analytically. For flash cooling in gas streams heat transfer is limited by the boundary layer, so that strains during cooling are small. Characteristic cooling times vary with crystal size L as L3/2. Plunge into liquids increases cooling rates by more than an order of magnitude. However, plunge cooling produces much larger internal temperature gradients and strains that may damage crystals, especially in the case of large crystals. Based on this analysis, factors affecting the success of flash cooling experiments can be ordered from most to least important as follows: crystal solvent content and solvent composition; crystal size and shape; amount of residual liquid around the crystal; cooling method (liquid or gas); choice of gas/liquid; relative speed between cooling fluid and crystal. Two problems related to flash cooling are investigated using X-ray imaging, diffraction peak shape measurements and conventional diffraction on tetragonal lysozyme. Step preceding flash cooling---post-growth treatment---is modelled by dehydration. The results indicate that near 88% relative humidity c lattice parameter decreases abruptly, mosaicity and diffraction resolution degrade sharply, topographs develop extensive contrast. This transformation exhibits metastability and hysteresis; original order can be restored by rehydration. These are features of the first order phase transition involving abrupt loss of crystal water. Consequently, post-growth treatment should either avoid lattice transitions or maintain the lattice in an ordered metastable state. Flash cooling of large (˜1 mm) crystals degrades diffraction resolution, broadens mosaicity and lattice spacings distribution. Lattice strain decreases and diffraction resolution improves with decreasing crystal size and with the usage of glycerol. Annealing reduces lattice constant spread and improves the resolution. X

  17. 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.

  18. 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.

  19. Liquid nitrogen dewar for protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Gaseous Nitrogen Dewar apparatus developed by Dr. Alex McPherson of the University of California, Irvine for use aboard Mir and the International Space Station allows large quantities of protein samples to be crystallized in orbit. The specimens are contained either in plastic tubing (heat-sealed at each end). Biological samples are prepared with a precipitating agent in either a batch or liquid-liquid diffusion configuration. The samples are then flash-frozen in liquid nitrogen before crystallization can start. On orbit, the Dewar is placed in a quiet area of the station and the nitrogen slowly boils off (it is taken up by the environmental control system), allowing the proteins to thaw to begin crystallization. The Dewar is returned to Earth after one to four months on orbit, depending on Shuttle flight opportunities. The tubes then are analyzed for crystal presence and quality

  20. Liquid nitrogen dewar for protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Gaseous Nitrogen Dewar apparatus developed by Dr. Alex McPherson of the University of California, Irvine for use aboard Mir and the International Space Station allows large quantities of protein samples to be crystallized in orbit. The specimens are contained either in plastic tubing (heat-sealed at each end). Biological samples are prepared with a precipitating agent in either a batch or liquid-liquid diffusion configuration. The samples are then flash-frozen in liquid nitrogen before crystallization can start. On orbit, the Dewar is placed in a quiet area of the station and the nitrogen slowly boils off (it is taken up by the environmental control system), allowing the proteins to thaw to begin crystallization. The Dewar is returned to Earth after one to four months on orbit, depending on Shuttle flight opportunities. The tubes then are analyzed for crystal presence and quality

  1. Relation between sequence and structure in membrane proteins.

    PubMed

    Olivella, Mireia; Gonzalez, Angel; Pardo, Leonardo; Deupi, Xavier

    2013-07-01

    Integral polytopic membrane proteins contain only two types of folds in their transmembrane domains: α-helix bundles and β-barrels. The increasing number of available crystal structures of these proteins permits an initial estimation of how sequence variability affects the structure conservation in their transmembrane domains. We, thus, aim to determine the pairwise sequence identity necessary to maintain the transmembrane molecular architectures compatible with the hydrophobic nature of the lipid bilayer. Root-mean-square deviation (rmsd) and sequence identity were calculated from the structural alignments of pairs of homologous polytopic membrane proteins sharing the same fold. Analysis of these data reveals that transmembrane segment pairs with sequence identity in the so-called 'twilight zone' (20-35%) display high-structural similarity (rmsd < 1.5 Å). Moreover, a large group of β-barrel pairs with low-sequence identity (<20%) still maintain a close structural similarity (rmsd < 2.5 Å). Thus, we conclude that fold preservation in transmembrane regions requires less sequence conservation than for globular proteins. These findings have direct implications in homology modeling of evolutionary-related membrane proteins. Supplementary data are available at Bioinformatics online.

  2. 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

  3. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1992-01-01

    A study is presented of the crystallization of isocitrate lyase (ICL) and the influence of the lack of thermal solutal convection in microgravity on the morphology of ICL crystals is discussed. The latest results of studies with thermonucleation are presented. These include the nucleation of a protein with retrograde solubility and an unknown solubility curve. A new design for a more microgravity compatible thermonuclear is presented.

  4. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%.

  5. Protein crystal growth - Growth kinetics for tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, M. L.; Snyder, R. S.; Naumann, R.

    1986-01-01

    Results are reported from theoretical and experimental studies of the growth rate of lysozyme as a function of diffusion in earth-gravity conditions. The investigations were carried out to form a comparison database for future studies of protein crystal growth in the microgravity environment of space. A diffusion-convection model is presented for predicting crystal growth rates in the presence of solutal concentration gradients. Techniques used to grow and monitor the growth of hen egg white lysozyme are detailed. The model calculations and experiment data are employed to discuss the effects of transport and interfacial kinetics in the growth of the crystals, which gradually diminished the free energy in the growth solution. Density gradient-driven convection, caused by presence of the gravity field, was a limiting factor in the growth rate.

  6. Protein crystallization - is it rocket science?

    PubMed

    DeLucas, L J.

    2001-07-01

    Fueled by initial space shuttle results, the National Aeronautics and Space Administration (NASA) has been supporting fundamental studies of macromolecular crystal growth since 1985. The majority of this research is directed at understanding the relationship between experimental variables and important crystal characteristics. The program has resulted in new methods and technology that will benefit the crystallography community's effort to meet the ever-increasing demand for protein structural information. Microgravity crystallization results indicate a potential impact on structural biology's more challenging problems, as soon as long-duration experiments can be performed on the International Space Station.

  7. Microscale vapour diffusion for protein crystallization.

    PubMed

    Korczyńska, Justyna; Hu, Ting Chou; Smith, David K; Jenkins, Joby; Lewis, Rob; Edwards, Tom; Brzozowski, Andrzej M

    2007-09-01

    The development of new crystallization platforms via the application of high-throughput technologies has delivered a plethora of crystallization plates suitable for robot-driven and manual setups. However, practically all these plates (except for microfluidic channel chips) are based on a very similar design and well (precipitant):drop (protein) volume ratios. A new type of crystallization plate (microplate) has therefore been developed and tested that still employs the classical vapour-diffusion technique but minimizes the precipitant well volume to 1.2 microl for a 150 nl protein drop setup. This enables a very significant saving on the total bulk of the crystallization screen, hence allowing the application of new, rare and expensive solutions in automated crystallization-screening procedures. Additionally, owing to the very low drop:well volume ratio, the new microplate can significantly accelerate the equilibrium time necessary for crystal nucleation and growth, in many cases shortening the high-throughput crystallization screening process to a few hours.

  8. Rotating Vessels for Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Cottingham, Paul

    2005-01-01

    Rotating vessels have been proposed as means of growing larger, more nearly uniform protein crystals than would otherwise be possible in the presence of normal Earth gravitation. Heretofore, nonrotating vessels have been used. It is difficult to grow high-quality protein crystals in the terrestrial gravitational field because of convection plumes created by the interaction between gravitation and density gradients in protein-solution depletion layers around growing crystals. The density gradients and the associated convection plumes cause the surfaces of growing crystals to be exposed to nonuniform solution densities, thereby causing the crystals to form in irregular shapes. The microgravitational environment of outer space has been utilized to eliminate gravitation-induced convection, but this approach is generally not favorable because of the high cost and limited availability of space flight. The use of a rotating vessel according to the proposal is intended to ameliorate the effects of gravitation and the resultant convection, relative to the corresponding effects in a non-rotating vessel. The rotation would exert an averaging effect over time, distributing the convective force on the depletion layer. Therefore, the depletion layer would be more nearly uniform and, as a result, the growing crystal would be more nearly perfect. The proposal admits of variations (see figure), including the following: The growing crystal could be rotated about its own central axis or an external axis. The crystal-growth vessel could be of any of various shapes, including cylindrical, hemispherical, conical, and combinations thereof. The crystal-growth vessel could be suspended in a viscous fluid in an outer vessel to isolate the growing crystal from both ambient vibrations and vibrations induced by a mechanism that drives the rotation. The rotation could be coupled to the crystal-growth vessel by viscous or magnetic means. The crystal-growth vessel could be supported within the

  9. Magnetic Control of Convection during Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Ramachandran, N.; Leslie, F. W.

    2004-01-01

    An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility

  10. Magnetic Control of Convection during Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Ramachandran, N.; Leslie, F. W.

    2004-01-01

    An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility

  11. 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.

  12. Characterization of the Outer Membrane Protein OprF of Pseudomonas aeruginosa in a Lipopolysaccharide Membrane by Computer Simulation

    SciTech Connect

    Straatsma, TP; Soares, Thereza A.

    2009-02-01

    The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded anti-parallel β-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared to simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared to the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.

  13. Nucleation and growth control in protein crystallization

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Nyce, Thomas A.; Meehan, Edward J.; Sowers, Jennifer W.; Monaco, Lisa A.

    1990-01-01

    The five topics summarized in this final report are as follows: (1) a technique for the expedient, semi-automated determination of protein solubilities as a function of temperature and application of this technique to proteins other than lysozyme; (2) a small solution cell with adjustable temperature gradients for the growth of proteins at a predetermined location through temperature programming; (3) a microscopy system with image storage and processing capability for high resolution optical studies of temperature controlled protein growth and etching kinetics; (4) growth experiments with lysozyme in thermosyphon flow ; and (5) a mathematical model for the evolution of evaporation/diffusion induced concentration gradients in the hanging drop protein crystallization technique.

  14. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    NASA Astrophysics Data System (ADS)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  15. 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.

  16. FNAS/advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1992-01-01

    A scintillation method is presented for determination of the temperature dependence of the solubility, S(T), of proteins in 50-100 micro-l volumes of solution. S(T) data for lysozyme and horse serum albumin were obtained for various combinations of pH and precipitant concentrations. The resulting kinetics and equilibrium information was used for dynamic control, that is the separation of nucleation and growth stages in protein crystallization. Individual lysozyme and horse serum albumin crystals were grown in 15-20 micro-l solution volumes contained in x-ray capillaries.

  17. Cry Protein Crystals: A Novel Platform for Protein Delivery

    PubMed Central

    Bonnegarde-Bernard, Astrid; Wallace, Julie A.; Dean, Donald H.; Ostrowski, Michael C.; Burry, Richard W.; Boyaka, Prosper N.; Chan, Michael K.

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues. PMID:26030844

  18. Cry protein crystals: a novel platform for protein delivery.

    PubMed

    Nair, Manoj S; Lee, Marianne M; Bonnegarde-Bernard, Astrid; Wallace, Julie A; Dean, Donald H; Ostrowski, Michael C; Burry, Richard W; Boyaka, Prosper N; Chan, Michael K

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

  19. Searching for the Best Protein Crystals: Synchrotron Based Measurements of Protein Crystal Quality

    NASA Technical Reports Server (NTRS)

    Borgstahl, Gloria; Snell, Edward H.; Bellamy, Henry; Pangborn, Walter; Nelson, Chris; Arvai, Andy; Ohren, Jeff; Pokross, Matt

    1999-01-01

    We are developing X-ray diffraction methods to quantitatively evaluate the quality of protein crystals. The ultimate use for these crystal quality will be to optimize crystal growth and freezing conditions to obtain the best diffraction data. We have combined super fine-phi slicing with highly monochromatic, low divergence synchrotron radiation and the ADSC Quantum 4 CCD detector at the Stanford Synchrotron Radiation laboratory beamline 1.5 to accurately measure crystal mosaicity. Comparisons of microgravity versus earth-grown insulin crystals using these methods will be presented.

  20. Searching for the Best Protein Crystals: Synchrotron Based Measurements of Protein Crystal Quality

    NASA Technical Reports Server (NTRS)

    Borgstahl, Gloria; Snell, Edward H.; Bellamy, Henry; Pangborn, Walter; Nelson, Chris; Arvai, Andy; Ohren, Jeff; Pokross, Matt

    1999-01-01

    We are developing X-ray diffraction methods to quantitatively evaluate the quality of protein crystals. The ultimate use for these crystal quality will be to optimize crystal growth and freezing conditions to obtain the best diffraction data. We have combined super fine-phi slicing with highly monochromatic, low divergence synchrotron radiation and the ADSC Quantum 4 CCD detector at the Stanford Synchrotron Radiation laboratory beamline 1.5 to accurately measure crystal mosaicity. Comparisons of microgravity versus earth-grown insulin crystals using these methods will be presented.

  1. Application of Membrane Crystallization for Minerals’ Recovery from Produced Water

    PubMed Central

    Ali, Aamer; Quist-Jensen, Cejna Anna; Macedonio, Francesca; Drioli, Enrico

    2015-01-01

    Produced water represents the largest wastewater stream from oil and gas production. Generally, its high salinity level restricts the treatment options. Membrane crystallization (MCr) is an emerging membrane process with the capability to extract simultaneously fresh water and valuable components from various streams. In the current study, the potential of MCr for produced water treatment and salt recovery was demonstrated. The experiments were carried out in lab scale and semi-pilot scale. The effect of thermal and hydrodynamic conditions on process performance and crystal characteristics were explored. Energy dispersive X-ray (EDX) and X-ray diffraction (XRD) analyses confirmed that the recovered crystals are sodium chloride with very high purity (>99.9%), also indicated by the cubic structure observed by microscopy and SEM (scanning electron microscopy) analysis. It was demonstrated experimentally that at recovery factor of 37%, 16.4 kg NaCl per cubic meter of produced water can be recovered. Anti-scaling surface morphological features of membranes were also identified. In general, the study provides a new perspective of isolation of valuable constituents from produced water that, otherwise, is considered as a nuisance. PMID:26610581

  2. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments.

    PubMed

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-09-01

    Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.

  3. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    PubMed Central

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. PMID:23999307

  4. Crystal nuclei templated nanostructured membranes prepared by solvent crystallization and polymer migration

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Ji, Jing; Li, Kang

    2016-09-01

    Currently, production of porous polymeric membranes for filtration is predominated by the phase-separation process. However, this method has reached its technological limit, and there have been no significant breakthrough over the last decade. Here we show, using polyvinylidene fluoride as a sample polymer, a new concept of membrane manufacturing by combining oriented green solvent crystallization and polymer migration is able to obtain high performance membranes with pure water permeation flux substantially higher than those with similar pore size prepared by conventional phase-separation processes. The new manufacturing procedure is governed by fewer operating parameters and is, thus, easier to control with reproducible results. Apart from the high water permeation flux, the prepared membranes also show excellent stable flux after fouling and superior mechanical properties of high pressure load and better abrasion resistance. These findings demonstrate the promise of a new concept for green manufacturing nanostructured polymeric membranes with high performances.

  5. Crystal nuclei templated nanostructured membranes prepared by solvent crystallization and polymer migration

    PubMed Central

    Wang, Bo; Ji, Jing; Li, Kang

    2016-01-01

    Currently, production of porous polymeric membranes for filtration is predominated by the phase-separation process. However, this method has reached its technological limit, and there have been no significant breakthrough over the last decade. Here we show, using polyvinylidene fluoride as a sample polymer, a new concept of membrane manufacturing by combining oriented green solvent crystallization and polymer migration is able to obtain high performance membranes with pure water permeation flux substantially higher than those with similar pore size prepared by conventional phase-separation processes. The new manufacturing procedure is governed by fewer operating parameters and is, thus, easier to control with reproducible results. Apart from the high water permeation flux, the prepared membranes also show excellent stable flux after fouling and superior mechanical properties of high pressure load and better abrasion resistance. These findings demonstrate the promise of a new concept for green manufacturing nanostructured polymeric membranes with high performances. PMID:27640994

  6. 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

  7. IR laser-induced protein crystal transformation

    SciTech Connect

    Kiefersauer, Reiner Grandl, Brigitte; Krapp, Stephan; Huber, Robert

    2014-05-01

    A novel method and the associated instrumentation for improving crystalline order (higher resolution of X-ray diffraction and reduced mosaicity) of protein crystals by precisely controlled heating is demonstrated. Crystal transformation is optically controlled by a video system. A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress.

  8. Membrane protein structure determination — The next generation☆☆☆

    PubMed Central

    Moraes, Isabel; Evans, Gwyndaf; Sanchez-Weatherby, Juan; Newstead, Simon; Stewart, Patrick D. Shaw

    2014-01-01

    The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. PMID:23860256

  9. Flexible photonic crystal membranes with nanoparticle high refractive index layers

    PubMed Central

    Paulsen, Moritz; Gerken, Martina

    2017-01-01

    Flexible photonic crystal slabs with an area of 2 cm2 are fabricated by nanoimprint replication of a 400 nm period linear grating nanostructure into a ≈60 µm thick polydimethylsiloxane membrane and subsequent spin coating of a high refractive index titanium dioxide nanoparticle layer. Samples are prepared with different nanoparticle concentrations. Guided-mode resonances with a quality factor of Q ≈ 40 are observed. The highly flexible nature of the membranes allows for stretching of up to 20% elongation. Resonance peak positions for unstretched samples vary from 555 to 630 nm depending on the particle concentration. Stretching results in a resonance shift for these peaks of up to ≈80 nm, i.e., 3.9 nm per % strain. The color impression of the samples observed with crossed-polarization filters changes from the green to the red regime. The high tunability renders these membranes promising for both tunable optical devices as well as visualization devices. PMID:28243558

  10. Flexible photonic crystal membranes with nanoparticle high refractive index layers.

    PubMed

    Karrock, Torben; Paulsen, Moritz; Gerken, Martina

    2017-01-01

    Flexible photonic crystal slabs with an area of 2 cm(2) are fabricated by nanoimprint replication of a 400 nm period linear grating nanostructure into a ≈60 µm thick polydimethylsiloxane membrane and subsequent spin coating of a high refractive index titanium dioxide nanoparticle layer. Samples are prepared with different nanoparticle concentrations. Guided-mode resonances with a quality factor of Q ≈ 40 are observed. The highly flexible nature of the membranes allows for stretching of up to 20% elongation. Resonance peak positions for unstretched samples vary from 555 to 630 nm depending on the particle concentration. Stretching results in a resonance shift for these peaks of up to ≈80 nm, i.e., 3.9 nm per % strain. The color impression of the samples observed with crossed-polarization filters changes from the green to the red regime. The high tunability renders these membranes promising for both tunable optical devices as well as visualization devices.

  11. 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

  12. 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

  13. 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.

  14. Sigmoid kinetics of protein crystal nucleation

    NASA Astrophysics Data System (ADS)

    Nanev, Christo N.; Tonchev, Vesselin D.

    2015-10-01

    A non-linear differential equation expressing the new phase nucleation rate in the different steps of the process (non-stationary and stationary nucleation and in the plateau region) is derived from basic principles of the nucleation theory. It is shown that one and the same sigmoid (logistic) function describes both nucleation scenarios: the one according to the classical theory, and the other according to the modern two-stage mechanism of protein crystal formation. Comparison to experimental data on both insulin crystal nucleation kinetics and on bovine β-lactoglobulin crystallization indicates a good agreement with the sigmoidal prediction. Experimental data for electrochemical nucleation and glass crystallization obey the same sigmoid time dependence, and suggest universality of this nucleation kinetics law.

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

    PubMed

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

    2017-07-20

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

  16. 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

  17. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1994-01-01

    The long-term stability of the interferometric setup for the monitoring of protein morphologies has been improved. Growth or dissolution of a crystal on a 100 A scale can now be clearly distinguished from dimensional changes occurring within the optical path of the interferometer. This capability of simultaneously monitoring the local interfacial displacement at several widely-spaced positions on the crystal surface with high local depth resolution, has already yielded novel results. We found with lysozyme that (1) the normal growth rate is oscillatory, and (2) the mean growth step density is greater at the periphery of a facet than in its center. The repartitioning of Na(+) and Cl(-) ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed to interpret the large body of data in a unified way. The results strongly suggests that (1) the ion to lysozyme ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter on the order of 10 microns. The computational model for diffusive-convective transport in protein crystallization (see the First Report) has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies. These results show that some elements of a moving boundary problem must be incorporated into the model in order to obtain a more realistic description. Our experimental setup for light scattering investigations of aggregation and nucleation in protein solutions has been extensively tested. Scattering intensity measurements with a true Rayleigh scatterer produced systematically increased forward scattering, indicating problems with glare. These have been resolved. Preliminary measurements with supersaturated lysozyme solutions revealed that the scatterers grow with time. Work has begun on a computer program

  18. 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.

  19. 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

  20. Helix-packing motifs in membrane proteins.

    PubMed

    Walters, R F S; DeGrado, W F

    2006-09-12

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

  1. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  2. (PCG) Protein Crystal Growth Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lyase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator for STS-26 was Charles Bugg.

  3. (PCG) Protein Crystal Growth Isocitrate Lysase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lysase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator on STS-26 was Charles Bugg.

  4. A Critical Evaluation of in silico Methods for Detection of Membrane Protein Intrinsic Disorder

    PubMed Central

    Pryor, Edward E.; Wiener, Michael C.

    2014-01-01

    Intrinsically disordered regions in proteins possess important biological roles including transcriptional regulation, molecular recognition, and provision of sites for posttranslational modification. In three-dimensional crystallization of both soluble and membrane proteins, identification and removal of disordered regions is often necessary for obtaining crystals possessing sufficient long-range order for structure determination. Disordered regions can be identified experimentally, with techniques such as limited proteolysis coupled with mass spectrometry, or computationally, by using disorder prediction programs, of which many are available. Although these programs use various methods to predict disorder from a protein’s primary sequence, they all were developed using information derived from soluble protein structures. Therefore, their performance and accuracy when applied to integral membrane proteins remained an open question. We evaluated the performance of 13 disorder prediction programs on a dataset containing 343 membrane proteins, and upon subdatasets containing only α-helical or β-barrel proteins. These programs were ranked using multiple metrics, including metrics specifically created for membrane proteins. Analysis of these data shows a clear distinction between programs that accurately predict disordered regions in membrane proteins and programs which perform poorly, and allows for the robust integration of in silico disorder prediction into our PSI:Biology membrane protein structural genomics pipeline. PMID:24739163

  5. Overexpression, Isolation, and Crystallization of Proteins

    NASA Astrophysics Data System (ADS)

    Skelly, Jane V.; Madden, C. Bernadette

    Rapid developments in recombinant technology have made it possible to overproduce selected proteins of specific interest to the levels required for structural analysis by X-ray crystallography. High-level gene expression has facilitated the purification of many proteins that are normally only expressed at low concentrations, as well as those that have proven difficult to purify to homogeneity from natural sources. Furthermore, advances in oligonucleotide site-directed mutagenesis have enabled proteins to be engineered so as to possess certain features that may confer stability or assist in then isolation. There are several examples of proteins that, despite rigorous purification from their natural source, have defied crystallization attempts, e.g., human growth hormone, but have been successfully crystallized from recombinant sources (1). The lack of posttranslational processing in bacterial expressed proteins can often be an advantage to the crystallographer where microheterogeneity presents a problem. Indeed, certain features or residues of a protein that are believed to impede crystal formation by preventing a close-packing arrangement may be successfully deleted by genetic manipulation without destroying its essential functionality (2).

  6. Effect of sludge retention on UF membrane fouling: The significance of sludge crystallization and EPS increase.

    PubMed

    Yu, Wenzheng; Graham, Nigel; Yang, Yunjia; Zhou, Zhiqi; Campos, Luiza C

    2015-10-15

    This paper concerns a previously unreported mechanism of membrane ultrafiltration (UF) fouling when a UF process with coagulation pre-treatment is used in drinking water treatment. The significance of settled coagulant solids (sludge) with different age within the membrane tank on UF fouling has been investigated at laboratory-scale, using model micro-polluted surface water. The process of floc crystallization and increasing bacterial EPS with solids (sludge) retention time may be detrimental to UF operation by causing an increased rate of membrane fouling. In this study the performance of two alum pre-treated hollow-fibre UF units, operated in parallel but with different settled sludge retention times (1 and 7 days), was compared. The results showed that over 34 days of operation the extent of reversible and irreversible fouling was much greater for the 7-day solids retention time. This was attributed to the greater extent of bacterial activity and the presence of Al-nanoparticles, arising from sludge crystallization, at the longer retention time. In particular, greater quantities of organic matter, particularly EPS (proteins and polysaccharides), were found in the UF cake layer and pores for the 7-day retention time. The addition of chlorine later in the membrane run substantially reduced the rate of membrane fouling for both sludge retention times, and this corresponded to reduced quantities of organic substances, including EPS, in the cake layer and pores of both membranes. The results suggest that bacterial activity (and EPS production) is more important than the production of Al-nanoparticles from solids crystallization in causing membrane fouling. However, it is likely that both phenomena are interactive and possibly synergistic.

  7. Fluorescence-Detectino Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins

    SciTech Connect

    Kawate,T.; Gouaux, E.

    2006-01-01

    Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.

  8. 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

  9. The Growth of Protein Crystals Using McDUCK

    NASA Technical Reports Server (NTRS)

    Ewing, Felicia; Wilson, Lori; Nadarajah, Arunan; Pusey, Marc

    1998-01-01

    Most of the current microgravity crystal growth hardware is optimized to produce crystals within the limited time available on orbit. This often results in the actual nucleation and growth process being rushed or the system not coming to equilibrium within the limited time available. Longer duration hardware exists, but one cannot readily pick out crystals grown early versus those which nucleated and grew more slowly. We have devised a long duration apparatus, the Multi-chamber Dialysis Unit for Crystallization Kinetics, or McDUCK. This apparatus-is a series of protein chambers, stacked upon a precipitant reservoir chamber. All chambers are separated by a dialysis membrane, which serves to pass small molecules while retaining the protein. The volume of the Precipitant chamber is equal to the sum of the volumes of the protein chamber. In operation, the appropriate chambers are filled with precipitant solution or protein solution, and the McDUCK is placed standing upright, with the precipitant chamber on the bottom. The precipitant diffuses upwards over time, with the time to reach equilibration a function of the diffusivity of the precipitant and the overall length of the diffusion pathway. Typical equilibration times are approximately 2-4 months, and one can readily separate rapid from slow nucleation and growth crystals. An advantage on Earth is that the vertical precipitant concentration gradient dominates that of the solute, thus dampening out solute density gradient driven convective flows. However, large Earth-grown crystals have so far tended to be more two dimensional. Preliminary X-ray diffraction analysis of lysozyme crystals grown in McDUCK have indicated that the best, and largest, come from the middle chambers, suggesting that there is an optimal growth rate. Further, the improvements in diffraction resolution have been better signal to noise ratios in the low resolution data, not an increase in resolution overall. Due to the persistently large crystals

  10. The Growth of Protein Crystals Using McDUCK

    NASA Technical Reports Server (NTRS)

    Ewing, Felicia; Wilson, Lori; Nadarajah, Arunan; Pusey, Marc

    1998-01-01

    Most of the current microgravity crystal growth hardware is optimized to produce crystals within the limited time available on orbit. This often results in the actual nucleation and growth process being rushed or the system not coming to equilibrium within the limited time available. Longer duration hardware exists, but one cannot readily pick out crystals grown early versus those which nucleated and grew more slowly. We have devised a long duration apparatus, the Multi-chamber Dialysis Unit for Crystallization Kinetics, or McDUCK. This apparatus-is a series of protein chambers, stacked upon a precipitant reservoir chamber. All chambers are separated by a dialysis membrane, which serves to pass small molecules while retaining the protein. The volume of the Precipitant chamber is equal to the sum of the volumes of the protein chamber. In operation, the appropriate chambers are filled with precipitant solution or protein solution, and the McDUCK is placed standing upright, with the precipitant chamber on the bottom. The precipitant diffuses upwards over time, with the time to reach equilibration a function of the diffusivity of the precipitant and the overall length of the diffusion pathway. Typical equilibration times are approximately 2-4 months, and one can readily separate rapid from slow nucleation and growth crystals. An advantage on Earth is that the vertical precipitant concentration gradient dominates that of the solute, thus dampening out solute density gradient driven convective flows. However, large Earth-grown crystals have so far tended to be more two dimensional. Preliminary X-ray diffraction analysis of lysozyme crystals grown in McDUCK have indicated that the best, and largest, come from the middle chambers, suggesting that there is an optimal growth rate. Further, the improvements in diffraction resolution have been better signal to noise ratios in the low resolution data, not an increase in resolution overall. Due to the persistently large crystals

  11. Influence of Crystal Expansion/Contraction on Zeolite Membrane Permeation

    SciTech Connect

    Sorenson, Stephanie G; Payzant, E Andrew; Noble, Richard D; Falconer, John L.

    2010-01-01

    X-ray diffraction was used to measure the unit cell parameters of B-ZSM-5, SAPO-34, and NaA zeolite powders as a function of adsorbate loading at 303 K, and in one case, at elevated temperatures. Most adsorbates expanded the zeolite crystals below saturation loading at 303 K: n-hexane and SF6 in B-ZSM-5, methanol and CO2 in SAPO-34, and methanol in NaA zeolite. As the loadings increased, the crystals expanded more. Changes in the unit cell volumes of B-ZSM-5 and SAPO-34 zeolite powders correlated with changes in permeation through zeolite membranes defects. When the zeolite crystals expanded or contracted upon adsorption, the defect sizes decreased or increased. In B-ZSM-5 membranes, the fluxes through defects decreased dramatically when n-hexane or SF6 adsorbed. In contrast, i-butane adsorption at 303 K contracted B-ZSM-5 crystals at low loadings and expanded them at higher loadings. Correspondingly, the flux through B-ZSM-5 membrane defects increased at low i-butane loadings and decreased at high loading because the defects increased in size at low loading and decreased at high loadings. At 398 K and 473 K, n-hexane expanded the B-ZSM-5 unit cell more as the temperature increased from 303 to 473 K. The silicalite-1 and B-ZSM-5 unit cell volumes expanded similarly upon n-hexane adsorption at 303 K; boron substitution had little effect on volume expansion.

  12. 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

  13. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1994-01-01

    A high-resolution microscopic interferometric setup for the monitoring of protein morphologies has been developed. Growth or dissolution of a crystal can be resolved with a long-term depth resolution of 200 A and a lateral resolution of 2 microns. This capability of simultaneously monitoring the interfacial displacement with high local depth resolution has yielded several novel results. We have found with lysozyme that (1) the normal growth rate is oscillatory, and (2) depending on the impurity content of the solution, the growth step density is either greater or lower at the periphery of a facet than in its center. The repartitioning of Na plus and Cl minus ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed, to interpret the large body of data in unified way. The results strongly suggest that (1) the ion to lysozyne ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter electron microscopy results appear to confirm this finding, which could have far-reaching consequences for x-ray diffraction studies. A computational model for diffusive-convective transport in protein crystallization has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies and our kinetics data for the growth of lysozyme. The results show that even in the small cell employed, protein concentration nonuniformities and gravity-driven solutal convection can be significant. The calculated convection velocities are of the same order to magnitude as those found in earlier experiments. As expected, convective transport, i.e., at Og, lysozyme crystal growth remains kinetically limited. The salt distribution in the crystal is predicted to be non-uniform at both 1g and 0g, as a consequence of protein depletion in the solution. Static and

  14. Membrane cholesterol access into a G-protein-coupled receptor

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  15. Membrane cholesterol access into a G-protein-coupled receptor

    PubMed Central

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-01-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs. PMID:28220900

  16. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    SciTech Connect

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  17. Low temperature heat capacity of phononic crystal membranes

    NASA Astrophysics Data System (ADS)

    Puurtinen, T. A.; Maasilta, I. J.

    2016-12-01

    Phononic crystal (PnC) membranes are a promising solution to improve sensitivity of bolometric sensor devices operating at low temperatures. Previous work has concentrated only on tuning thermal conductance, but significant changes to the heat capacity are also expected due to the modification of the phonon modes. Here, we calculate the area-specific heat capacity for thin (37.5 - 300 nm) silicon and silicon nitride PnC membranes with cylindrical hole patterns of varying period, in the temperature range 1 - 350 mK. We compare the results to two- and three-dimensional Debye models, as the 3D Debye model is known to give an accurate estimate for the low-temperature heat capacity of a bulk sample. We found that thin PnC membranes do not obey the 3D Debye T3 law, nor the 2D T2 law, but have a weaker, approximately linear temperature dependence in the low temperature limit. We also found that depending on the design, the PnC patterning can either enhance or reduce the heat capacity compared to an unpatterned membrane of the same thickness. At temperatures below ˜ 100 mK, reducing the membrane thickness unintuitively increases the heat capacity for all samples studied. These observations can have significance when designing calorimetric detectors, as heat capacity is a critical parameter for the speed and sensitivity of a device.

  18. 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.

  19. 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

  20. Interferometer Protein Crystal Growth (IPCG) system

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This view shows a large growth cell. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

  1. Interferometer Protein Crystal Growth (IPCG) system

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This view shows the optical bench. The growth cell would be on the right side. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

  2. Interferometer Protein Crystal Growth (IPCG) system

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This diagram shows the growth cells. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

  3. Interferometer Protein Crystal Growth (IPCG) system

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This diagram shows the optical layout. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

  4. Interferometer Protein Crystal Growth (IPCG) system

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This view shows interferograms produced in ground tests. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

  5. The Effect of Protein Impurities on Lysozyme Crystal Growth

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Forsythe, Elizabeth L.; Pusey, Marc L.

    1998-01-01

    While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. In order to further examine the issue of purity in macromolecule crystallization this study investigates the effect of the protein impurities, avidin, ovalbumin and conalbumin, at concentrations up to 50%, on the solubility, crystal face growth rates and crystal purity, of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the f {101} and {101} lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit ope