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Studien zur Proteintranslokation in Escherichia coli : Untersuchung der Membranproteine SecYEG und YidC unter Verwendung biochemischer und kristallographischer Methoden

By Mirko Lotz

Abstract

Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.Der Transport von Proteinen in zelluläre Membranen hinein oder durch diese hindurch wird durch die konservierte und überall anzutreffende Sec-Maschinerie vermittelt. Das Sec-Homolog in der inneren Membran von Escherichia coli ist SecYEG. Der Sec-vermittelte Einbau von vielen Membranproteinen wird unterstützt von YidC, einem weiteren Protein in der inneren Membran von Escherichia coli. YidC führt zusätzlich den Einbau einiger Proteine Sec-unabhängig durch. YidC gehört zu einer konservierten aber strukturell uncharakterisierten Familie von Proteinen, die wichtig für die Biogenese von Membranproteinen sind und hat Homologe in Mitochondrien und Chloroplasten. Durch Modifikation eines früheren Kristallisations-Protokolles wurde zweidimensionale Kristalle von SecYEG in Gegenwart des Signalsequenz-Peptides von LamB gezüchtet. Das Aufnehmen von Strukturdaten mittels Elektronen-Kryomikroskopie und die Berechnung einer Differenzstruktur, welche eine frühere SecYEG-Projektionsstruktur mit der von SecYEG kristallisiert in Gegenwart von Substrat vergleicht, demonstrierte eine Reihe neuer bzw. fehlender Dichten. Diese deuten auf das Binden von Signalpeptid in räumlicher Nähe zur Translokationspore sowie auf signifikante Umlagerungen in der Nähe des lateralen Austrittsortes für Transmembrandomänen in SecYEG hin. Die Differenzstruktur legt nahe, dass dimeres SecYEG ein asymmetrisches Molekül ist, bestehend aus einem aktiven und einem inaktiven SecYEG-Monomer. Detergenzentfernung aus einem Gemisch von gereinigtem YidC und Lipiden brachte zweidimensionale Kristalle hervor, welche bezüglich ihrer Bildung sehr abhängig von Ionenstärke und Lipidkomposition waren. Eine elektronen-kryomikroskopische Untersuchung der in hydratisiertem Zustand gefrorenen Kristalle und Bildverarbeitung machten strukturelle Details mit einer Auflösung von etwa 10 Å sichtbar. Mitteln von Einzelbildern zu zwei alternativen Projektionsstrukturen in p2 bzw. p121_a Symmetrie brachte grundsätzlich gleiche strukturelle Merkmale hervor. Vier YidC-Monomere bilden eine Einheitszelle (Dimensionen 82 x 71 Å, eingeschlossener Winkel 85 ° bzw. 90 °) und scheinen als zwei Dimere vorzuliegen, welche in gegensätzlicher Orientierung in die Membran eingebettet sind. Ein Bereich geringer Dichte im Zentrum eines jeden YidC-Monomers stellt möglicherweise eine Einstülpung der Membran dar, die besondere Bedeutung für den Einbau von Substratproteinen in die Lipid-Doppelschicht haben könnte

Topics: Proteinsekretion, Proteintransport, Proteine, Membranproteine, Membran, Kryokonservierung, Kryoelektronenmikroskopie, Kristallzüchtung, ddc:570
Year: 2008
OAI identifier: oai:publikationen.ub.uni-frankfurt.de:319

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