The formation and insertion of OmpA β -barrel domain into the lipid bilayers take place by a concerted mechanism

How the mechanism of strands association in pairs is related with the mechanism of OmpA barrel insertion into lipid bilayers was an important issue addressed by the present study. The characteristics of strands association in pairs were analyzed in the previous sections: 5.5.1 to 5.5.3. In the section 5.5.3, I have shown that the strands association in pairs and the translocation across the lipid bilayer are two concerted processes.

In the present section I describe the concerted mechanism of barrel assembly and translocation across the lipid bilayer. The structure of this mechanism is based on the previous studies concerning the OmpA barrel translocation across the lipid bilayer (Kleinschmidt et al 1999; Kleinschmidt and Tamm 1999) and on the findings of the present study, which has investigated the process of barrel assembly. The concerted mechanism of barrel formation and insertion into lipid bilayer is summarized in Figure 5.8 and Table 5.6. According with this mechanism the barrel assembly and insertion into the lipid bilayer is a sequence of three steps, which precedes the formation of the native OmpA structure.

During the first step (Figure 5.8, A and Table 5.6, A) the unfolded OmpA, is absorbed at the membrane surface and a first intermediate (IM₁) is formed (Kleinschmidt et al 1999;

Kleinschmidt and Tamm 1999). Previously was show that most of β-structure (but not yet the β-barrel) is formed upon OmpA absorption to membrane surface (Surrey and Jähnig 1992;

Rodionova et al. 1995).

My results indicate that an incipient strands association characterizes the intermediate IM1

(Figure 5.8, A and Table 5.6, A). Kleinschmidt et al 1999 suggested that this first intermediate would appear as a series of connected, but independently foldedβ-hairpins, arranged in a star like fashion or like the ribs in an umbrella. The degrees of fluorescence quenching (Table 5.6, A) are consistent with this view of IM1. The top-end of β2-β3, β1-β2and β1-β8pairs appear to display the most efficient degree of association which fact is consistent with the early insertion of the barrel top end into the bilayer.

In the next stage of folding (represented by the intermediate IM2) the top-end of β3 (W57) inserts first into lipid bilayer, followed closely by the top end of β₁ (W15). The degree of association of β₂-β₃,β₁-β₂andβ₁-β₈in IM₁is consistent with the way in which, they insert in the bilayer during the formation of IM2. The strands penetrate superficially the lipid bilayer during the second step that leads to the formation of IM₂. All the tryptophan residues are located at very similar distances from the bilayer centre (around 10Ǻ) (Kleinschmidt et al 1999). Clearly this intermediate displays an advanced stage of strands assembly but the barrel does not appear to be completely formed yet (Figure 5.8, B and Table 5.6, B).

The difference between strands association at bottom end and top end of barrel is reduced in comparison with IM₁, but still significant. Thus, the bottom end of barrel marked by W7 displays a weaker strands association. In contrast, the top end is involved in a more effective strands assembly (Figure 5.8, B and Table 5.6, B). This difference between the top end and bottom end of OmpA barrel observed in the case of IM₁and IM₂is triggered by the fact that the top end of strandsβ1,β2and β3(marked by the residues W15 and W57) insert first into the bilayer (Kleinschmidt et al 1999).

The last intermediate IM₃, which precedes the formation of the native OmpA, appears to present a barrel almost completely formed (Figure 5.8, A and Table 5.6, C) with the residues marking the top end of OmpA barrel in contact with the centre of bilayer, inserted deep in the hydrophobic core of bilayer (Kleinschmidt et al 1999). The strands association and formation of IM3is a result of interaction of the polypeptide chains with the hydrophobic core of bilayer.

This interaction requires the polar residues to form hydrogen bonds. Kleinschmidt et al 1999 suggested that IM contains a significant number of hydrogen bonds.

Finally, complet strands association characterizes the native OmpA form (Figure 5.8, D and Table 5.6, D). The fluorescence quenching values obtained at 40°C (Figure 5.8, D and Table 5.6, D) confirm that the OmpA barrel is fully formed (see also Figure 5.7, C). Stable hydrogen bonding is expected to take place at this stage of folding. The formation of stable hydrogen bonds is confirmed by the appearance of the 30 kDa form of OmpA.

Figure 5.8 Scheme for the concerted mechanism of β-strands association and barrel insertion of OmpA. The β-strands are on average -~45° inclined from the membrane normal (Rodionova et al 1995; Pautsch and Schulz 1998). The pairs of Trp (Wn) and Cys (Cm) residues choosen to monitor the strands association are represented as coloured circles. The colour code is indicated in Table 5.6 (see below). The folding model displays a sequence of three intermediates preceding the appartition of native OmpA. This folding mechanism shows the nearly synchronous association and membrane insertion ofβ-strands.A, during the first step of folding (IM1) OmpA is absorbed on membrane surface and the strands are loosely associated (Table 5.6, A).B, in the second step (IM2) the strands penetrate slightly the bilayer and associate more efficiently (Table 5.6, B). C ,in the third step (IM3) the top end of barrel is in contact with the centre of bilayer (W15, W57, L162, L35) the barrel is nearly formed (Table 5.6,C).D, the OmpA is in the native (N) form (Table 5.6, D).

Table 5.6 Values of tryptophan fluorescence quenching characterizing the intermediates formed during OmpA folding.

FQ/F(1 h)^c

A^d B^e C^f D^g

Wn^a Cm^b (IM1) (IM2) (IM3) (N)

●

^W15(β1)

●

^C35(β2) 0.69 0.48 0.47 0.41

●

^W15(β1)

●

^C162(β8) 0.72 0.45 0.38 0.30

●

^W57(β3)

●

^C35(β2) 0.65 0.47 0.49 0.40

●

^W7(β1)

●

^{C43 (β2) 0.92 0.59 0.46 0.35}

●

^{W7 (β}1)

●

^C170(β8) 0.87 0.57 0.53 0.37

aTryptophan residue on β_n strand; ^bCysteine residue on β_m strand; The association process between strandsβ_nandβ_mtriggers the quenching of Wn fluorescence emission by spin-labelled Cm;

cValues of Wn fluorescence quenching F_Q/F (1 h) (taken from Table 5.5); ^d-gIntermediates formed during strands association and OmpA folding;

5.6 Conclusions

I have developed a novel approach to study the association of neighbouringβ-strands in pairs during formation of OMPsβ-barrel. This new method is based on monitoring the fluorescence quenching of tryptophan emission by spin-labelled cysteines.

As a result of applying this new technique, I have observed that (i) the β-strands of OmpA associate in pairs nearly synchronous and (ii) the OmpA barrel formation and insertion into membrane are two very closely correlated processes.

Based on my results and on previous studies I have proposed a multi-step concerted mechanism of OmpA folding, which covers the OmpA barrel assembly and the insertion into the lipid bilayer (Figure 5.8).

Summary

The mechanism of protein folding represents a major challenge for the modern biology. While considerable knowledge and insight has been obtained on the folding of globular water-soluble proteins the folding and membrane insertion of integral membrane proteins (IMPs) is far less well understood and also not as extensively investigated due to the difficulties with handling this particular category of proteins.

Integral membrane proteins fall into two different classes that can be distinguished according to their secondary structure: α-helical andβ-barrel proteins. The subject of the present thesis is bacterial 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.

My work focussed on several significant aspects of the folding mechanism of OmpA. In the first project, I analysed the effect of the phospholipid composition of model membranes (lipid bilayers) on the folding kinetics of outer membrane protein A. My second and my third project describe two parallel pathways of the chaperone-assisted folding of OmpA. In the fourth project I further dissected the assembly of the β-barrel domain in relation to its membrane insertion. In the following paragraphs, I summarize the most important findings of my studies and how they correlate with previously reported results.

Phospholipids are essential components for the folding and insertion process of outer membrane proteins (OMPs) due to the fact that OMPs have to be integrated into the outer membrane (OM), which is composed of phospholipids in the inner leaflet and of lipopolysaccharide in the outer leaflet. All previously published studies on the role of phospholipids on the folding and insertion of OmpA were carried out in vitro using model membranes composed of phosphatidylcholines (PCs), which are not natural components of the OM of E.coli. 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 technique employed in this project was: kinetics of tertiary structure formation determined by electrophoresis (KTSE), an SDS-PAGE based method used to analyze the folding kinetics of OMPs. The results obtained during the investigations 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 tendency of PE to form nonbilayer structures or 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. Best results were obtained with (i) lipid bilayers composed of PC, PG and PE at molar ratios of 50, 20 and 30, respectively and (ii) model membranes containing PC and PG at molar ratios of 80 to 20. When PG was included in moderate amounts (20-30%) into PC/PG membranes, the folding kinetics of OmpA were stimulated significantly. 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. Thus, the insertion of OmpA from the aqueous phase into the hydrophobic core of the lipid bilayer is facilitated for membranes containing PG.

My next two projects sought to explore possible chaperone assisted folding pathways of OmpA that may exist in bacteria. Previous studies suggested that periplasmic proteins like the Seventeen kDa Protein (Skp) and the survival factor A (SurA) assist the assembly of OMPs into the outer membrane. First, I investigated the effects of Skp and LPS on the folding of OmpA into lipid bilayers in in vitroexperiments. In a subsequent project, the chaperone effect of SurA on OmpA folding was analyzed. I could demonstrate that both periplasmic chaperones improved folding of OmpA independent of each other.

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. Here, 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 ofn= 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

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. KTSE, fluorescence spectroscopy and circular dichroism were the methods used to explore the effects of Skp and LPS on OmpA folding and insertion into lipid bilayers.

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 chaperone effect of SurA on OmpA folding increases with the SurA concentration and becomes maximal at a molar ratio of 2 SurA per 1 OmpA.

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. Previous studies showed that the transmembrane segments of the 8-stranded β-barrel domain of OmpA inserts into the lipid bilayer in a concerted fashion and that the rate of formation of natively folded OmpA and the rate of secondary structure formation are very similar. 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. In this new approach, I used a series of single Trp, single Cys mutants of OmpA. The Trp and Cys residues of each mutant were located in 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. Fluorescence spectroscopy was used to monitor the changes in fluorescence emission of these OmpA mutants.

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 individualβ-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 OmpAβ-barrel domain into the lipid bilayers is concerted. The investigations further confirmed that there are at least three membrane-bound folding intermediates of OmpA, which were previously described.

Zusammenfassung

Der Auflärung der Mechanismen der Proteinfaltung ist eine große Herausforderung für die moderne Biologie. Während über die Faltung von globulären, löslichen Proteinen bereits zahlreiche wissenschaftliche Studien publiziert wurden, ist die Faltung von integralen Membranproteinen (IMPs) bislang weit weniger intensiv untersucht worden. Dies liegt unter anderem daran, dass Membranproteine sehr viel schwieriger zu handhaben sind. Es gibt zwei Klassen von integralen Membranproteinen, die sich in der Sekundärstruktur ihrer Transmembrandomänen unterscheiden: die α-Helix Bündel- und die β -Fass-Membranproteine. 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. OmpA dient als Model in der Erforschung der Faltung und des Membraneinbaus von Aussenmembranproteinen (OMPs).

Ich habe den Mechanismus der Faltung von OmpA in Bezug auf mehrere Aspekte untersucht.

Mein erstes Forschungsprojekt beinhaltete die Untersuchung der Bedeutung der Phospholipide für die Geschwindigkeit der Faltung (d.h. ihres Einflusses auf die Faltungskinetiken) von OmpA in Lipiddoppelschichten. Das zweite und das dritte Forschungsprojekt beinhalteten Untersuchungen zu zwei parallelen, Chaperone-vermittelteten Faltungswegen von OmpA. Das vierte Projekt war eine Untersuchung über die Faltung des β -Fasses von OmpA in Bezug auf den Einbau in die Membran. Die wichtigsten Ergebnisse meiner Studien, habe ich in den folgenden Abschnitten zusammengefasst.

Phospholipide sind essentiell für den Faltungsmechanismus der Aussenmembranproteine, weil sie Hauptbestandteile der inneren Monoschicht der Lipid-Doppelschicht der Aussenmembran (OM) sind. In früheren Untersuchungen zur Rolle der Phospholipide beim Einbau und der Faltung von OmpA wurden Phosphatidylcholine (PCs) verwendet, die jedoch keine natürlichen Komponenten der OM sind. 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). Als Methode habe ich die KTSE (Kinetik der Bildung von Tertiär-Struktur mittels Elektrophorese)-Technik verwendet. Das ist eine Methode, die zur Auswertung der Kinetik die Natriumdodecylsulfat-Polyacrylamidgelelektrophorese

(SDS-PAGE) benutzt. 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 beste Faltungseffizienz wurde entweder mit (i) Lipiddoppelschichten aus PC, PG und PE im Molverhältnis 50, 20 und 30, oder aber mit (ii) Membranen aus PC und PG im Molverhältnis von 80 zu 20 erreicht. 20-30% PG in der Membran haben die Faltung von OmpA signifikant stimuliert. 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 zweiin vivomöglicherweise parallele, Chaperone-vermittelte Faltungswege von OmpA untersucht. Früher war gezeigt worden, dass die Deletionen der Gene der periplasmatischen Proteine Skp und SurA jeweils zu reduzierten Konzentrationen von Außenmembranproteinen in der OM von E. coli führen. Da sich das Gen von Skp auf dem Chromosom in unmittelbarer Nachbarschaft zu Genen befindet, die an der Biosynthese von Lipopolysaccharid (LPS) beteiligt sind, und LPS außerdem ein Hauptbestandteil der Außenmembran von Gram-negativen Bakterien ist, habe ich LPS in diese Untersuchungen miteinbezogen.

Zuerst habe ich die Auswirkungen von Skp und LPS auf den Einbau und die Faltung von OmpA in Lipiddoppelschichten untersucht. Anschließend wurde die Auswirkung der Chaperone SurA auf die Faltung von OmpA studiert. Genetische Studien an E. coli hatten postuliert, dass SurA und Skp an zwei parallelen, Chaperone-vermittelteten Faltungswegen von OmpA beteiligt sind. Ich habe hier gezeigt, dass beide Chaperone die Faltung von OmpA in Lipid-Doppelschichten unabhängig voneinander beschleunigen.

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. Als Methoden wurden in dieser Studie die Fluoreszenzspektroskopie, die Circulardichroismusspektroskopie und das

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. Als Methoden wurden in dieser Studie die Fluoreszenzspektroskopie, die Circulardichroismusspektroskopie und das

Im Dokument Kinetic Studies on the Folding and Insertion of Outer Membrane Protein A from Escherichia Coli (Seite 125-137)