Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli

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2007
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Bulieris, Paula Vasilichia
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Kinetische Studien über die Faltung und Membraninsertion des Außenmembranproteins A von Escherichia Coli
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Zusammenfassung

My work focussed on several significant aspects of the folding mechanism of outer membrane protein A of Escherichia coli, which is composed of a 155 residue periplasmic domain and of a 170 residue transmembrane (TM) domain that forms an 8-stranded TM β-barrel.
First, I investigated the folding kinetics of OmpA into model membranes containing the main components of the inner leaflet of the OM: phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The results obtained showed that model membranes mimicking the composition of the outer membrane (containing PE and PG at a molar ratio of 80 and 20) resulted in very low OmpA folding efficiency possibly due to the strong surface-dehydration caused by intermolecular hydrogen-bonds between the ammonium- and the phosphate- parts of PE headgroups. In contrast model membranes where PE was excluded or partially replaced with PC (which forms bilayers and contains a charged trimethyl ammonium group which cannot participate in hydrogen bonding) resulted in high folding yields. When PG was included in moderate amounts (20-30%) into PC/PG membranes, the folding kinetics of OmpA were stimulated significantly because the repulsion between negatively charged PG molecules and the increased hydration shell of this headgroup leads to more water in the headgroup region of negatively charged PG compared to PC.
My next two projects sought to explore possible chaperone assisted folding pathways of OmpA that may exist in bacteria. In bacteria, outer membrane proteins like OmpA are synthesized in the cytosol. They are then translocated across the cytoplasmic (inner) membrane into the periplasm in unfolded form. Periplasmic chaperones like SurA or Skp bind to the polypeptide chains after they emerge from the translocon.
I demonstrated that OmpA binds ~ 3 molecules of Skp and forms a soluble complex in which OmpA is kept largely unfolded. This complex then binds a small number of n = 2-7 LPS per OmpA in solution to form a folding and insertion component form of OmpA that is bound to Skp and LPS. In this second complex, OmpA develops no or only very small amounts of secondary structure. When this insertion competent form of OmpA was reacted with preformed phospholipid bilayers, OmpA rapidly inserted and folded to its native state. The results indicated that OmpA that is bound to Skp and LPS does not fold in absence of lipid bilayers, but folds into lipid bilayers with accelerated folding kinetics. In contrast, the kinetics of OmpA folding into lipid bilayers from a state bound to Skp in absence of LPS or from a denatured state in 8 M urea were both slower.
The SurA assisted folding pathway of OmpA is markedly different in comparison with the folding pathway described above. According to my results, LPS is not required and the presence of SurA prevents the interaction of OmpA with LPS. The chaperone effect of SurA was manifested in experiments where OmpA was incubated in aqueous buffer either in absence or in presence of SurA. In absence of SurA, preincubation in aqueous solution led to aggregated forms of OmpA that did not fold upon subsequent addition of lipid bilayers. In presence of SurA, the formation such aggregated forms was suppressed. SurA therefore appeared to prevent aggregation of OmpA, which still folded into lipid bilayers in high yields. In contrast to experiments with Skp and LPS, for which folding was stimulated compared to simple refolding experiments of urea-denatured OmpA into lipid bilayers, the effect of SurA on unfolded OmpA was small.
The last chapter of my thesis presents relevant new data concerning the mechanism of formation and membrane insertion of transmembrane β-barrel domain of OmpA. The goal of my study was to investigate the formation of the transmembrane β-barrel domain of OmpA (residues 1 to 171) on the level of individual β-strands and to relate their association to the insertion of OmpA into the lipid membrane.
A new method was developed to monitor the association of individual β-strands in pairs during the formation of the β-barrel domain. I used a series of single Trp, single Cys mutants of OmpA. The Trp and Cys residues of each mutant were located on neighbouring β-strands. The Cysteine-residue was labelled with a nitroxyl spin-label that functions as a short-range fluorescence quencher. Association of the neighboring β-strands during OmpA folding triggers the quenching of Trp fluorescence signal by the spin-labelled Cys residue.
The results of the last chapter of the present thesis led to the following conclusions: (i) the assembly of individual strands in pairs during the OmpA barrel formation is a correlated, highly concerted process (and not a sequential one), (ii) the association of individual β-strands in pairs and the sealing of the 8 stranded β-barrel between strands 1 and 8 take place simultaneously (iii) the mechanism of formation and insertion of OmpA β-barrel domain into the lipid bilayers is concerted.

Zusammenfassung in einer weiteren Sprache

Das Außenmembranprotein A (OmpA) von Escherichia coli, dessen Faltung in dieser Dissertation studiert wurde, besteht aus einer periplasmatischen und aus einer Transmembrandomäne, die in der äußeren Membran eine 8-strängige β-Fass-Struktur ausbildet. Ich habe den Mechanismus der Faltung von OmpA in Bezug auf mehrere Aspekte untersucht.
Ich habe die Faltungskinetiken von OmpA in Lipiddoppelschichten untersucht, die die Haupt-Lipidkomponenten der inneren Monoschicht der Aussenmembran enthielten, nämlich Phosphatidylethanolamin (PE) und Phosphatidylglycerol (PG). Die Ergebnisse dieses Projektes haben gezeigt, dass Lipiddoppelschichten, die die gleiche Lipidzusammensetzung besitzen wie die OM (nämlich PE und PG, im Molverhältnis von 80 zu 20), zu einer sehr niedrigen Faltungseffizienz von OmpA führen. Die starke Tendenz von PE, hexagonale Phasen invertierter Mizellen zu bilden oder aber die starke Dehydratation der Membranoberfläche von PE sind mögliche Gründe für diese Beobachtung. Im Gegensatz dazu wurden bessere Faltungsausbeuten erzielt, wenn PE entfernt wurde oder zumindest teilweise durch PC (das keine hexagonalen Phasen invertierter Mizellen ausbildet) ersetzt wurde. Die elektrostatische Abstoßung zwischen negativ geladenen PG Molekülen und der größere Hydratationsmantel dieser Kopfgruppe resultiert in einem höheren Wassergehalt der Kopfgruppenregion von Lipidmembranen aus Phosphatidylglycerol im Vergleich zu Membranen des zwitterionischen Phosphatidylcholin. Daher ist es wahrscheinlich, dass die Insertion von OmpA von der wässerigen Phase in die hydrophobe Region der Lipid-Doppelschicht für Membranen aus PG weniger Aktivierungsenergie benötigt und schneller ist.
In zwei meiner weiteren Projekte habe ich zwei in vivo möglicherweise parallele, Chaperone-vermittelte Faltungswege von OmpA untersucht.
In Bakterien werden Außenmembranproteine wie OmpA im Cytosol synthetisiert. Sie werden dann in entfalteter Form durch das Translokon SecYEG über die Cytoplasmamembran in das Periplasma gebracht. Periplasmatische Chaperone wie SurA oder Skp binden die entfalteten Polypeptidketten, nachdem zuvor eine Peptidase die Signalsequenz der OMPs abgespalten hat.
Hier habe ich in vitro gezeigt, dass entfaltetes OmpA drei Moleküle Skp bindet und ein löslicher Komplex ausgebildet wird. Dieser Komplex bindet etwa 3-7 Moleküle LPS in Lösung. OmpA bildet im Komplex mit Skp bzw. Skp und LPS etweder keine oder nur geringfügige Anteile an Sekundärstruktur aus, faltet aber aus diesem Komplex bei Zugabe von Lipid-Doppelschichten. OmpA faltet aus diesem ternären Komplex bei pH 7 mit höheren Faltungsausbeuten und schnelleren Faltungskinetiken in Phospholipid-Doppelschichten, als dies für entfaltetes OmpA in 8 M Harnstofflösung, oder aber für OmpA in Anwesenheit von nur einer Komponente, entweder Skp oder LPS, beobachtet wurde.
Der SurA assisitierte Faltungsweg von OmpA unterschied sich von dem beschriebenen Skp/LPS assistierten Faltungsweg deutlich. Nach meinen Ergebnissen ist die Gegenwart von LPS für den SurA-assistierten Faltung von OmpA nicht notwendig. Die Gegenwart von SurA verhinderte die verzögernde Wirkung von LPS (in Abwesenheit von Skp) auf den Membraneinbau von OmpA. Die Untersuchungen zeigten, dass SurA die Aggregation von OmpA zurückdrängt, aber nicht direkt auf entfaltetes OmpA wirkt.
Das letzte Projekt meiner Dissertationsarbeit ergab neue wichtige Informationen zum Faltungsprozess von OmpA, insbesondere zur Ausbildung der β-Fass-Struktur der Transmembrandomäne. Frühere Studien hatten gezeigt, dass die Transmembransegmente des 8-strängigen β­-Fasses synchronisiert in die Lipiddoppelschicht inserieren und dass die Kinetiken der Ausbildung von Sekundär- und Tertiärstruktur gleiche Geschwindigkeits­konstan­ten aufweisen. Das Ziel meiner Studie war es, die Ausbildung der β-Fass-Struktur (Reste 1-171) auf der Ebene einzelner β-Stränge zu untersuchen, und die Assoziation der β-Stränge mit dem Membraninsertionsprozess zu korrelieren.
Dazu habe ich eine neue Methode entwickelt, die es erlaubte, die Assoziation der β-Stränge zeitaufgelöst zu verfolgen. Ich habe dazu 5 Mutanten präpariert, die jeweils einen einzelnen Tryptophan- und einen einzelnen Cysteinrest enthielten (Einzel-Trp-einzel-Cys-Mutanten). In jedem dieser 5 Mutanten, waren der Tryptophan und der Cystein-Rest in benachbarten β-Strängen positioniert. Vor dem Faltungsexperiment wurde der Cysteinrest mit einem Spin-Label markiert, dass als Fluoreszenzquencher diente und die Fluoreszenz im gefalteten Zustand der OmpA-Mutante löschte.
Diese Messungen führten zu folgenden Ergebnissen: (i) Die Assoziation individueller β-Stränge zu antiparallelen β-Strangpaaren war korreliert und erfolgte nicht sequentiell. (ii) Die Assoziation von benachbarten β-Strängen und der Zusammenschluss des β-Fasses zwischen Strang β1 und Strang β8 erfolgte synchronisiert. (iii) die Ausbildung der β-Fass-Struktur war an den Membraneinbau gekoppelt.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Proteinfaltung, ß-Fass-Struktur, OmpA, Skp, SurA, LPS, protein folding, ß-barrel domain
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Zitieren
ISO 690BULIERIS, Paula Vasilichia, 2007. Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Bulieris2007Kinet-8829,
  year={2007},
  title={Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli},
  author={Bulieris, Paula Vasilichia},
  address={Konstanz},
  school={Universität Konstanz}
}
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In contrast model membranes where PE was excluded or partially replaced with PC (which forms bilayers and contains a charged trimethyl ammonium group which cannot participate in hydrogen bonding) resulted in high folding yields. When PG was included in moderate amounts (20-30%) into PC/PG membranes, the folding kinetics of OmpA were stimulated significantly because the repulsion between negatively charged PG molecules and the increased hydration shell of this headgroup leads to more water in the headgroup region of negatively charged PG compared to PC.&lt;br /&gt;My next two projects sought to explore possible chaperone assisted folding pathways of OmpA that may exist in bacteria. In bacteria, outer membrane proteins like OmpA are synthesized in the cytosol. They are then translocated across the cytoplasmic (inner) membrane into the periplasm in unfolded form. Periplasmic chaperones like SurA or Skp bind to the polypeptide chains after they emerge from the translocon.&lt;br /&gt;I demonstrated that OmpA binds ~ 3 molecules of Skp and forms a soluble complex in which OmpA is kept largely unfolded. This complex then binds a small number of n = 2-7 LPS per OmpA in solution to form a folding and insertion component form of OmpA that is bound to Skp and LPS. In this second complex, OmpA develops no or only very small amounts of secondary structure. When this insertion competent form of OmpA was reacted with preformed phospholipid bilayers, OmpA rapidly inserted and folded to its native state. The results indicated that OmpA that is bound to Skp and LPS does not fold in absence of lipid bilayers, but folds into lipid bilayers with accelerated folding kinetics. In contrast, the kinetics of OmpA folding into lipid bilayers from a state bound to Skp in absence of LPS or from a denatured state in 8 M urea were both slower.&lt;br /&gt;The SurA assisted folding pathway of OmpA is markedly different in comparison with the folding pathway described above. According to my results, LPS is not required and the presence of SurA prevents the interaction of OmpA with LPS. The chaperone effect of SurA was manifested in experiments where OmpA was incubated in aqueous buffer either in absence or in presence of SurA. In  absence of SurA, preincubation in aqueous solution led to aggregated forms of OmpA that did not fold upon subsequent addition of lipid bilayers. In presence of SurA, the formation such aggregated forms was suppressed. SurA therefore appeared to prevent aggregation of OmpA, which still folded into lipid bilayers in high yields. 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December 18, 2007
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