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  1. Article ; Online: Thinking outside the crystal: complementary approaches for examining transporter conformational change.

    Elvington, Shelley M / Maduke, Merritt

    Channels (Austin, Tex.)

    2008  Volume 2, Issue 5, Page(s) 373–379

    Abstract: As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of ... ...

    Abstract As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present ¹⁹F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.
    MeSH term(s) Membrane Transport Proteins/chemistry ; Molecular Probe Techniques ; Protein Conformation
    Chemical Substances Membrane Transport Proteins
    Language English
    Publishing date 2008-12-08
    Publishing country United States
    Document type Journal Article ; Review
    ZDB-ID 2262854-X
    ISSN 1933-6969 ; 1933-6950
    ISSN (online) 1933-6969
    ISSN 1933-6950
    DOI 10.4161/chan.2.5.6903
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs.

    Elvington, Shelley M / Nichols, J Wylie

    Biochimica et biophysica acta

    2007  Volume 1768, Issue 3, Page(s) 502–508

    Abstract: ... Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight ...

    Abstract It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k(dis)=0.115 s(-1); t(1/2)=6.03 s); Bodipy FL-PC (k(dis)=5.2x10(-4); t(1/2)=22.2 min); Bodipy 530-PC (k(dis)=1.52x10(-5); t(1/2)=12.6 h); and Bodipy 581-PC (k(dis)=5.9x10(-6); t(1/2)=32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.
    MeSH term(s) Azoles ; Fluorescent Dyes ; Fluorometry ; Kinetics ; Molecular Structure ; Nitrobenzenes ; Phosphatidylcholines/chemistry ; Phosphatidylcholines/metabolism
    Chemical Substances 7-nitrobenz-2-oxa-1,3-diazol-4-yl ; Azoles ; Fluorescent Dyes ; Nitrobenzenes ; Phosphatidylcholines ; 1-palmitoyl-2-oleoylphosphatidylcholine (TE895536Y5)
    Language English
    Publishing date 2007-03
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 60-7
    ISSN 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    ISSN (online) 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
    ISSN 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    DOI 10.1016/j.bbamem.2006.11.013
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  3. Article ; Online: Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR.

    Elvington, Shelley M / Liu, Corey W / Maduke, Merritt C

    The EMBO journal

    2009  Volume 28, Issue 20, Page(s) 3090–3102

    Abstract: The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that ... ...

    Abstract The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.
    MeSH term(s) Antiporters/chemistry ; Chloride Channels/chemistry ; Escherichia coli Proteins/chemistry ; Magnetic Resonance Spectroscopy ; Models, Molecular ; Protein Structure, Secondary
    Chemical Substances Antiporters ; Chloride Channels ; ClC protein, E coli ; Escherichia coli Proteins ; hydrogen-chloride symporter
    Language English
    Publishing date 2009-09-10
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 586044-1
    ISSN 1460-2075 ; 0261-4189
    ISSN (online) 1460-2075
    ISSN 0261-4189
    DOI 10.1038/emboj.2009.259
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  4. Article ; Online: Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.

    Khantwal, Chandra M / Abraham, Sherwin J / Han, Wei / Jiang, Tao / Chavan, Tanmay S / Cheng, Ricky C / Elvington, Shelley M / Liu, Corey W / Mathews, Irimpan I / Stein, Richard A / Mchaourab, Hassane S / Tajkhorshid, Emad / Maduke, Merritt

    eLife

    2016  Volume 5

    Abstract: CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its ... ...

    Abstract CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
    MeSH term(s) Chloride Channels/chemistry ; Chloride Channels/metabolism ; Crystallography, X-Ray ; Electron Spin Resonance Spectroscopy ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/metabolism ; Magnetic Resonance Spectroscopy ; Models, Molecular ; Molecular Dynamics Simulation ; Protein Conformation
    Chemical Substances Chloride Channels ; ClC protein, E coli ; Escherichia coli Proteins
    Language English
    Publishing date 2016-01-22
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.11189
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  5. Article: Fluorescent, acyl chain-labeled phosphatidylcholine analogs reveal novel transport pathways across the plasma membrane of yeast.

    Elvington, Shelley M / Bu, Fang / Nichols, J Wylie

    The Journal of biological chemistry

    2005  Volume 280, Issue 49, Page(s) 40957–40964

    Abstract: Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although ...

    Abstract Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.
    MeSH term(s) 4-Chloro-7-nitrobenzofurazan/analogs & derivatives ; 4-Chloro-7-nitrobenzofurazan/metabolism ; ATP-Binding Cassette Transporters ; Acylation ; Adenosine Triphosphatases/physiology ; Adenosine Triphosphate/analysis ; Adenosine Triphosphate/physiology ; Biological Transport ; Boron Compounds ; Cell Membrane/metabolism ; Fluorescent Dyes ; Membrane Transport Proteins/physiology ; Phosphatidylcholines/metabolism ; Proton-Motive Force ; Saccharomyces cerevisiae/growth & development ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae/ultrastructure ; Saccharomyces cerevisiae Proteins/physiology
    Chemical Substances 1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine ; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene ; ATP-Binding Cassette Transporters ; Boron Compounds ; Fluorescent Dyes ; Lem3 protein, S cerevisiae ; Membrane Transport Proteins ; Phosphatidylcholines ; Saccharomyces cerevisiae Proteins ; Adenosine Triphosphate (8L70Q75FXE) ; Adenosine Triphosphatases (EC 3.6.1.-) ; Dnf2 protein, S cerevisiae (EC 3.6.1.3) ; Dnf1 protein, S cerevisiae (EC 7.6.2.1) ; 4-Chloro-7-nitrobenzofurazan (EQF2794IRE)
    Language English
    Publishing date 2005-10-04
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.M507926200
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  6. Article ; Online: Revealing an outward-facing open conformational state in a CLC Cl–/H+ exchange transporter

    Chandra M Khantwal / Sherwin J Abraham / Wei Han / Tao Jiang / Tanmay S Chavan / Ricky C Cheng / Shelley M Elvington / Corey W Liu / Irimpan I Mathews / Richard A Stein / Hassane S Mchaourab / Emad Tajkhorshid / Merritt Maduke

    eLife, Vol

    2016  Volume 5

    Abstract: CLC secondary active transporters exchange Cl- for H+. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its ... ...

    Abstract CLC secondary active transporters exchange Cl- for H+. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using 19F NMR, we show that as [H+] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
    Keywords antiporter ; membrane exchanger ; crystallization ; principal component analysis ; double electron-electron resonance spectroscopy ; membrane protein ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 500
    Language English
    Publishing date 2016-01-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: A designed inhibitor of a CLC antiporter blocks function through a unique binding mode.

    Howery, Andrew E / Elvington, Shelley / Abraham, Sherwin J / Choi, Kee-Hyun / Dworschak-Simpson, Sierra / Phillips, Sabrina / Ryan, Christopher M / Sanford, R Lea / Almqvist, Jonas / Tran, Kevin / Chew, Thomas A / Zachariae, Ulrich / Andersen, Olaf S / Whitelegge, Julian / Matulef, Kimberly / Du Bois, Justin / Maduke, Merritt C

    Chemistry & biology

    2012  Volume 19, Issue 11, Page(s) 1460–1470

    Abstract: The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC "Chloride Channel" family represents a unique target for biophysical ...

    Abstract The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC "Chloride Channel" family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of a specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4'-octanamidostilbene-2,2'-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid dependence of inhibition suggest potential mechanisms of action. This compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders.
    MeSH term(s) Antiporters/antagonists & inhibitors ; Antiporters/chemistry ; Antiporters/genetics ; Antiporters/metabolism ; Binding Sites ; Chloride Channels/metabolism ; Escherichia coli Proteins/antagonists & inhibitors ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Lipid Bilayers ; Molecular Dynamics Simulation ; Mutation ; Stilbenes/metabolism ; Stilbenes/pharmacology ; Sulfonic Acids/metabolism ; Sulfonic Acids/pharmacology
    Chemical Substances 4,4'-octanamidostilbene-2,2'-disulfonic acid ; Antiporters ; CLC-1 channel ; CLC-ec1 protein, E coli ; Chloride Channels ; Escherichia coli Proteins ; Lipid Bilayers ; Stilbenes ; Sulfonic Acids
    Language English
    Publishing date 2012-11-21
    Publishing country United States
    Document type Journal Article ; Research Support, American Recovery and Reinvestment Act ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 917827-2
    ISSN 1879-1301 ; 1074-5521
    ISSN (online) 1879-1301
    ISSN 1074-5521
    DOI 10.1016/j.chembiol.2012.09.017
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