Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism

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2007
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Baladhandapani, Shanmugavadivu
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Titel in einer weiteren Sprache
Die spontane Faltung von menschlichem VDAC in Lipidmembranen ist orientiert und beinhaltet einen teilweise unabhängigen Mechanismus
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Zusammenfassung

The entire thesis work presented here concerns the structure, folding and assembly of human mitochondrial outer membrane protein, VDAC and the major outcome of this thesis was summarized in separate sections below.
In the first study, initially a protocol for isolation of hVDAC1 in pure form with high yield was established. Next, the hVDAC1 purified in denatured form was successfully refolded to its native conformation and a single-step folding protocol for hVDAC1 was established. During the refolding trials, several important observations were made and the findings are listed below;
1. Folding of hVDAC1 into lipid bilayers was spontaneous and did not require any proteinaceous machinery or energy in the form of ATP to assist folding. This would list hVDAC1 as the first eukaryotic -barrel to spontaneously fold into lipid bilayers.
2. Also the result implied the earlier finding for soluble proteins that the fold of a protein appears to be solely encoded in the primary amino acid sequence of the protein and this was found to be applicable even to membrane proteins of human origin provided the conditions are not denaturing.
3. An interesting and important outcome from this study was that correct-folding of hVDAC1 occurred only in lipid bilayers and not in detergent micelles. Mild detergents like LDAO, DDM and C8E4 commonly used for solubilizing and crystallizing purposes did not allow proper folding of hVDAC1 and this required subsequent reconstitution into membranes.
4. hVDAC1 was found to always insert in one direction into the membrane and the finding concluded oriented membrane insertion for hVDAC1.
In the next study, the mechanism behind and steps involved in folding and membrane insertion of hVDAC1 were explored. In this study, the kinetics of membrane insertion was analyzed mainly by fluorescence spectroscopy. The results revealed some similarities and several notable differences from OmpA, the existing well established model for prokaryotic β-barrel. The major conclusions are listed below;
1. hVDAC1 was largely folded in aqueous buffer and thus further folding and membrane insertion had less restrictive requirements (listed in next points). Further, kinetics of folding and membrane insertion was faster when compared with bacterial β-barrels.
2. Most obvious outcome was membrane insertion of hVDAC1 into phosphatidylcholine bilayers like diC12:0PC and diC14:0PC had a low energy barrier and required only low activation energy.
3. Membrane insertion of hVDAC1 was found to proceed via two membrane-associated intermediates, first a membrane-adsorbed and second a partially membrane-inserted intermediate.
4. Folding and insertion was found to take place independent of the lipid concentration but dependent on lipid bilayer properties like membrane thickness and head group.
5. Folding and insertion was found to be sequential and occurred in separate steps. All the above results indicated strong differences in the mechanism and folding pathway for hVDAC1 when compared with bacterial β-barrels. The work also established hVDAC1 as a first model of eukaryotic origin to study the principles of folding and assembly of β-barrel membrane proteins.
In the third study, an attempt was made to probe the structure / the transmembrane topology of hVDAC1 in lipid bilayer. The study needed further experiments to analyze the topology. Nevertheless it yielded valuable information regarding the location of a tryptophan residue which is disputed until recently.
1. It was found that the tryptophan-209 has a membrane location and inserted into the lipid bilayer. To our knowledge the study was the first experimental proof to confirm the location of tryptophan in hVDAC1 and differed from the existing 13 β-strand model (Colombini 2004) on hVDAC1.
2. In addition, the work showed that the intramolecular distance between the -helix and last β-strand in the barrel was at least greater than 15-20 Å. The results would be helpful and be a necessary input for future structural studies on VDAC.
In the final study, the effect of lipid bilayer thickness and flexibility was investigated in detail on the example of OmpA. Since membrane thickness was found to largely affect insertion of β-barrel proteins (Kleinschmidt and Tamm 2002a), this study was conducted. The study showed that flexibility, surface curvature and elastic moduli of the lipid affect the activation energy of insertion of proteins.

Zusammenfassung in einer weiteren Sprache

In der vorliegenden Dissertations wurde der Mechanismus des Membraneinbaus und der Faltung eines eukaryotischen Außenmembranproteins untersucht, dass nach allen vorliegenden Informationen wie die bakteriellen Außenmembranproteine eine β-Fass Transmembranstruktur ausbildet. Die hier vorliegende Dissertation behandelt die Struktur, die Faltung, und den Einbau des humanen, spannungsabhängigen mitochondrialen Anionen-selektiven Kanals (VDAC, Voltage-Dependent Anion-selective Channel) in Lipidmembranen. Eine Reihe von Studien wurden dazu durchgeführt, die in einzelnen Abschnitten separat zusammengefasst sind.
In der ersten Studie habe ich zunächst ein Protokoll für die Reinigung und Isolation des humanen VDAC, isoform 1 (hVDAC1) implementiert und dann ein Einzelschritt-Rückfaltungsprotokoll in Lipid-Doppelschichten mit hohen Ausbeuten entwickelt. Während der Faltungsexperimente wurden eine Reihe wichtiger Beobachtungen gemacht:
1. Die Faltung des hVDAC1 zu seiner funktionell aktiven Form war spontan und weder Proteinfaltungshelfer oder Faltungsmaschinerie noch Energie¬liefe¬ran¬ten wie ATP waren für die Faltung erforderlich. hVDAC1 ist das erste eukaryotische Protein, das spontan in Lipid-Doppelschichten faltet, die als Modellmembranen dienen.
2. Die Resultate zeigten auch, dass das früher für lösliche Proteine gefundene Prinzip, dass die 3-D Struktur eines Proteins durch seine Primärstruktur festgelegt ist, auch für Membranproteine eukaryotischer Membranen wie hVDAC1 gilt, solange die Umgebung des Proteins nicht-denaturierend ist.
3. Die korrekte Faltung von hVDAC1 fand in meinen Experimenten nur in Lipid-Doppelschichten statt, nicht aber in Detergens-Mizellen. Milde Detergenzien wie LDAO, DDM, und C8E4, die weit verbreitet sind und für Solubilisierung sowie für strukturelle und funktionelle Untersuchungen zum Einsatz kommen, waren für die korrekte Faltung des hVDAC1 nicht ausreichend. Es war stattdessen notwendig, eine nachfolgende Rekonstitution aus Detergenzmicellen in Membranen durchzuführen, um die korrekte Sekundärstruktur zu erhalten.
4. Die direkte Insertion und Faltung des hVDAC1 in Membranen resultierte in nur einer Orientation dieses Kanals in Lipid-Doppelschichten.
5. In Einzelkanal-Leitfähigkeitsmessungen wurde die Spannungsabhängigkeit der Leitfähigkeit von zurückgefaltetem hVDAC1 demonstriert.

In einer zweiten Studie habe ich den Mechanismus und die Stufen der Faltung und des Einbaus von hVDAC1 in Lipidmembranen mittels Fluoreszenzspektroskopie, Circulardichroismusspektroskopie und Pepsin-katalysierter Proteolyse untersucht. Die Ergebnisse zeigen, dass für hVDAC1 die Faltung und die Insertion zu einem großen Teil entkoppelt sind und im Gegensatz zu dem für bakterielles Außenmembranprotein A (OmpA) gefundenen Mechanismus große Teile der Sekundärstruktur bereits vor der Membraninsertion ausgebildet werden. Für OmpA wurde hingegen zuvor eine starke Kopplung der Ausbildung von Sekundärstruktur an den Membraneinbau beobachtet. Die wesentlichen Beobachtungen waren:
1. Die Sekundärstruktur des hVDAC1 war zu einem sehr großen Teil bereits in der wässerigen Phase ausgebildet. Deshalb waren die Bedingungen für den nachfolgenden Einbau in eine Membran weniger restriktiv als für bakterielles OmpA. Die beobachteten Faltungskinetiken waren auch entsprechend schneller für hVDAC1 verglichen zu OmpA aus E. coli.
2. Der Membraneinbau und die Faltung von hVDAC1 in Phosphatidylcholin Lipid-Doppelschichten war für diC12:0PC und diC14:0PC durch eine niedrige Aktivierungsenergie charakterisiert.
3. Der Membraneinbau von hVDAC1 erfolgte unabhängig von der Lipid-Konzentration über zwei Membran-gebundene Intermediate von denen das erste Membran-assoziert war und das zweite offenbar etwas tiefer in die Membran inseriert war.
4. Die Faltung und der Membraneinbau waren in einer Minuten-Zeitskala unabhängig von der Lipid-Konzentration, jedoch abhängig von der Struktur der Lipide (Kettenlänge, Art der polaren Kopfgruppe) und den physikalischen Eigenschaften der Lipid-Doppelschicht.
5. Sowohl Faltung als auch Insertion fanden in mehreren Schritten statt und waren teilweise entkoppelt, was einen Unterschied zu den bisher studierten bakteriellen Membranproteinen wie OmpA darstellt. Diese Studie etablierte VDAC als das erste mitochondriale und das erste menschliche Membranprotein, für das die Prinzipien des Einbaus und der Faltung untersucht werden können.
In der 3. Studie habe ich weitere Eigenschaften der Struktur und der Transmembrantopologie in Lipiddoppelschichten untersucht und interessante Informationen über die Position von Tryptophanresten des hVDAC1 in der Membran erhalten, die bisher zur Diskussion standen

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Membranprotein Faltung, VDAC, Membrane Protein Folding, VDAC, Outer membrane protein
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ISO 690BALADHANDAPANI, Shanmugavadivu, 2007. Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Baladhandapani2007Spont-8665,
  year={2007},
  title={Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism},
  author={Baladhandapani, Shanmugavadivu},
  address={Konstanz},
  school={Universität Konstanz}
}
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