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Membrane proteins are a diverse group of proteins that serve a multitude of purposes with one of the most important ones being transport. All kinds of substrates are shuffled over biological membranes with the help of dedicated proteins enabling the transport along and against a concentration gradient. Within the group of actively transporting proteins a diverse set of proteins that rely on an electrochemical gradient to facilitate transport of a substrate against its concentration gradient can be found. Those so-called secondary active
transporters are a group on integral membrane proteins ubiquitous to all cells. They allow the transport of all kinds of substrates like nutrients, ions, other metabolites and drugs over the hydrophobic barrier created by the cellular and organellar membrane. The gradients that provide the main driving force for most of the transporters are either sodium ions or protons, although transporters utilizing other ions or organic compounds are found as well. In case of exchangers two very similar substrates are transported in opposing direction over the membrane, one against its electrochemical gradient driven by the other.
Along with a structural diversity of the transporters concerning overall shape, oligomerization and number of transmembrane elements comes a mechanistic variety though still following the principle of alternating access. In humans the malfunction of secondary active transporters can lead to a physiological disorders such as epilepsy, depression or obesity.
The focus of this thesis was the structural and functional characterization of the secondary active transporter SeCitS from Salmonella enterica, a symporter of the 2-hydroxycarboxylate family. The transport of citrate as a bivalent ion is facilitated by the flux of sodium ions that have an inward-facing gradient over the inner membrane of Salmonella enterica. Transport experiments showed that the transport ratio is two sodium ions per citrate molecule, netting in an electroneutral transport. Compared to other members of the family the specificity of the transporter towards its main substrate is very high.
Structural information on the protein was initially obtained through 2D electron crystallography, which allowed the identification of the oval shaped dimer and a first hint towards a significant conformational change that the protein undergoes during its transport cycle. Using 3D crystallography, the X-ray structure of the transporter was solved. The protein crystalizes as a stable, but conformationally asymmetric dimer. As bound citrate can be readily identified in both protomers they can be assigned into an outward- and an inward-facing conformation, with the main citrate binding site in the outward-facing conformation.
One interesting feature of the crystal structure was the large surface available for multimerization, providing a platform for tight dimerization of the two protomers. On the other hand, SeCitS did not show a true cooperativity of transport. With those two aspects taken into account the question arose if any potential crosstalk between the monomers within the dimer takes place and influences transport (negative cooperativity) or the conformational distribution within the dimer (stabilization of the protein within the membrane).
The functional approach in answering this question was the use of mutated variants of the protein for cross-linking within one monomer. Two residues were chosen respectively to lock one of either conformation to be able to test for transport activity in the remaining protomer. The suitability of the residues was derived from the crystal structure (D112 – R205 to lock the inward-facing conformation and L337 – S412 for the outward-facing conformation). After initial promising results the final variants were not stable enough to be analyzed in transport assays.
To analyze the distribution of relative conformations within the dimer the protein was reconstituted into native-like lipid environment such as nanodiscs or saposin nanoparticles to be analyzed by cryo-electron microscopy. The first images were recorded and did yield promising 2D classes where the general features of the transporter were identified. Yet, an improved preparation is required to obtain a high resolution structure.
The key functional aspects of a transporter are its ability to bind and transport its substrates. In a set of experiments those features were investigated by a radioligand transport assay and by isothermal titration calorimetry (ITC). The transport properties of the protein were assessed in a filter assay using a radioactively labeled citrate as a read-out. The protein was reconstituted into proteoliposomes and subjected to different substrate conditions. Different ions were tested in its ability to drive or inhibit transport, but only sodium ions were able to drive transport and also not hindered by the presence of other ions...
Diese Arbeit ist ein detaillierter Bericht über die Forschungsaktivitäten, die ich während meiner Promotion am Max-Planck-Institut für Biophysik durchgeführt habe. Mit dem Aufkommen der direkten Elektronendetektoren erlebte die Transmissionselektronenmikroskopie von gefrorenen hydratisierten Proben (Kryo-EM) einen epochalen Wandel, die sogenannte “Auflösungsrevolution”. Ab den 2010er Jahren ermöglichte die Kommerzialisierung der ersten direkten Detektoren die Erforschung biologischer Phänomene in beispiellosem Detail und machte Kryo-EM zu einer der leistungsstärksten (und gefragtesten) Forschungsmethoden in den Biowissenschaften. Meine Forschung konzentrierte sich auf die Verwendung der Elektronen-Kryotomographie, um zwei herausfordernde Ziele zu erreichen. Das erste bestand darin, die Denaturierung von Proteinen an der Luft-Wasser-Grenzfläche zu untersuchen, und das zweite die molekulare Landschaft eines lichtempfindlichen Chloroplastenvorläufers, des Etioplasten, zu beschreiben. Um die Relevanz, Herausforderungen und Auswirkungen meiner Arbeit zu vermitteln, habe ich diese Arbeit in drei Kapitel unterteilt.
Kapitel eins enthält eine Einführung in die Transmission-Elektronenmikroskopie.
Nach einer kurzen Zusammenfassung der historischen Meilensteine in der Disziplin beschreibe ich die wesentlichen Komponenten des TEM und deren Funktionsweise. Hier lege ich besonderen Wert auf die Struktur elektromagnetischer Linsensysteme, wie sie den Weg der Elektronen beim Durchlaufen der Säule beeinflussen und wie Bilder entstehen. Der hardwarebezogene Teil der Einführung wird durch eine vereinfachte Beschreibung der Elektronendetektoren abgeschlossen, in der ich die revolutionären Aspekte der direkten Elektronendetektoren, mit der Struktur und Funktion von CCD-Detektoren (Charge Coupled Device detector) vergleiche. Als nächstes konzentriere ich mich auf die theoretischen Prinzipien der Bilderzeugung. Um die Hauptphänomene im Zusammenhang mit der Bildqualität in TEM hervorzuheben, stelle ich grundlegende Konzepte wie den Einfluss von Elektronenenergie und optischen Aberrationen vor, gefolgt von einer ausführlicheren Beschreibung des Ursprungs von Kontrast und Rauschen. Der Unterabschnitt schließt mit einigen Überlegungen darüber, wie - und vor allem wie effizient - Detektoren kontinuierliche Elektronenwellen in diskrete Bereiche (Pixel) abtasten. Der folgende Unterabschnitt ist der Erfassung und Verarbeitung tomografischer Daten gewidmet. Hier gebe ich eine vereinfachte Beschreibung, wie Kippserien mit dem Mikroskop erfasst werden und wie die Rohdaten zu einer dreidimensionalen Darstellung der Probe verarbeitet werden. Der Einfluss der Neigungsgeometrie und der Dosisverteilung auf die Rekonstruktionsqualität wird ebenfalls diskutiert. Der zweite Teil des Unterabschnitts befasst sich mit der Strukturbestimmung durch Subtomogramm-Mittelung und der Errechnung der Auflösung von Kryo-EM-Rekonstruktion. Zuletzt schließe ich das Kapitel mit einer Beschreibung der Vorbereitung biologischer Proben für die Kryo-EM-Bildgebung mit einigen abschließenden Bemerkungen zur Dynamik und den Grenzen der Vitrifizierung ab.
Kapitel zwei folgt dem Thema der Kryo-Präparation biologischer Proben mit der Untersuchung der Denaturierung von Proteinen an der Luft-Wasser-Grenzfläche.
Im Einführungsabschnitt skizziere ich die wichtigsten Aspekte dieses Phänomens. Frühe Experimente zum Verhalten von Proteinen in Lösung zeigten ihre Neigung, aus der Lösung zu ihrer Grenzfläche mit der Atmosphäre zu diffundieren. Hier bilden sie meist unlösliche Schichten denaturierter Fibrillen Es wurde vorgeschlagen, dass die Korrelation zwischen Proteindenaturierung und Kontakt mit der Grenzfläche auf einen allmählichen Entfaltungsprozess zurückzuführen ist, bei dem Tausende von Wechselwirkungen pro Sekunde zu einer immer größeren strukturellen Schädigung führen würden. Ein direkter Beweis für diesen Mechanismus wurde jedoch nie dokumentiert. Um einen tieferen Einblick in die Dynamik an der Luft-Wasser-Grenzfläche zu erhalten, sammelte ich Kryotomogramme vitrifizierter Präparate der Fettsäuresynthase (FAS, Fatty Acid Synthase) aus Hefe. Im ersten Unterabschnitt der Ergebnisse beschreibe ich, wie die biochemische und Negativkontrastierung-TEM-Analyse von FAS-Fraktionen zeigte, dass der Komplex während des gesamten Reinigungsverfahrens intakt und katalytisch aktiv blieb. Nach der Vitrifizierung ergab die Einzelpartikelanalyse jedoch, dass 90% aller Komplexe stark beschädigt waren. Die tomographische Rekonstruktion derselben Proben zeigte, dass alle FAS-Komplexe an die Luft-Wasser-Grenzfläche gebunden waren. Die Seite des Moleküls, die der Grenzfläche ausgesetzt war, schien abgeflacht zu sein, während die Seite, in der wässrigen Phase, ihre native Struktur beibehielt. Die Mittelung der Subtomogramme bestätigte, dass eine Seite von fast 90% der Partikel stark beschädigt war. Durch den Vergleich der Ausrichtung dieser beschädigten Seite mit der Position eines Rechenmodells der Luft-Wasser-Grenzfläche konnte ich nachweisen, dass sie perfekt übereinstimmen, was den ersten direkten Beweis dafür liefert, dass die Wechselwirkung mit der Luft-Wasser-Grenzfläche die lokale Denaturierung großer Proteinkomplexe herbeiführt.
...
Electron microscopy (EM) demarcates itself from other structural biology techniques by its applicability to a large range of biological objects that spans from whole cells to individual macromolecules. In single-particle cryo-EM, frozen-hydrated samples, prepared by vitrification with liquid ethane, retain macromolecules in a medium that approximates their natural aqueous environment and that, in this way, preserves high-resolution structural information. Nonetheless, the sensitivity of biological specimens to the high-energy electron beam introduces restrictions on the total dose that can be used during imaging while avoiding significant radiation damage. Consequently, the signal-to-noise ratio attained in each individual image is very low, and structures with high-resolution detail must be recovered by averaging thousands of projections in random orientations. This is achieved through the use of image processing algorithms capable of aligning and classifying particle images through the evaluation of cross-correlation functions between each particle and a reference.
In recent years, several innovations took place in the field of single-particle cryo-EM, among which the development of direct electron detectors must be highlighted. Direct electron detectors have a better detective quantum efficiency (DQE) than both photographic film and CCD cameras, and offer a fast readout, compatible with the acquisition of movie stacks. Additionally, new image processing software has become available, with more sophisticated algorithms and designed to take advantage of the specific characteristics of the movies produced with direct electron detectors. These technological advances in both hardware and software catalyzed a revolution in single-particle cryo-EM, which is now routinely used for the determination of near-atomic structures. As a result, the range of macromolecules accessible to cryo-EM has increased drastically, as targets that were unsuitable before for imaging due to their small dimensions can now be adequately visualized and refined to high-resolution.
During my doctoral work, I have used single-particle cryo-EM to structurally characterize challenging membrane proteins, with a strong emphasis on protein complexes from aerobic respiratory chains. In chapter I of this thesis, I present my results on the bovine respirasome, a mitochondrial supercomplex composed of complexes I, III and IV. Chapter II is dedicated to the analysis of the structure of alternative complex III (ACIII) from Rhodothermus marinus, a bacterial quinol:cytochrome c/HiPIP oxidoreductase unrelated to the canonical cytochrome bc1 complex (complex III). In addition, in chapter III I describe the structure of KimA, a high-affinity potassium transporter that drives the transport of its substrate by using the energy stored in the form of a proton gradient. These three membrane proteins, with molecular weights ranging from 140 kDa to 1.7 MDa, illustrate the possibilities and limitations faced in single-particle cryo-EM.
The aerobic respiratory chain is responsible for the generation of a transmembrane difference of electrochemical potential that is then used by ATP synthase for the production of ATP or for driving solute transport over the membrane. They catalyze the transfer of electrons from a substrate, such as NADH or succinate, to molecular oxygen and use the chemical energy released in these redox reactions to drive the translocation of protons, or in some cases sodium ions, to the intermembrane space in mitochondria or the periplasm in bacteria.
In mitochondria, the respiratory chain is composed of four complexes: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). While it was for a long time believed that these complexes existed as single entities in the membrane, the use of milder procedures for protein purification and analysis revealed that respiratory complexes associate into well-ordered structures, known as supercomplexes. These have been proposed to offer different structural and functional advantages that are still controversial, including substrate channeling, stabilization of individual complexes and reduction of reactive oxygen species (ROS) production. The most thoroughly studied respiratory supercomplex has been the respirasome, conserved in higher eukaryotes and composed of one copy of complex I, a complex III dimer and one complex IV. By single-particle cryo-EM analysis, I retrieved a 9 Å map of the respirasome from Bos taurus, which allowed the accurate docking of atomic models of the three component complexes. The structure shows that complex III associates to the concave side of the membrane arm of complex I, while complex IV is located between the end of the complex I hydrophobic arm and complex III. Several defined protein-protein contacts are observed between the component complexes, which are mediated predominantly by supernumerary subunits and close to the membrane surfaces. The interactions established between complex I and complex III are extensive and may support the argument that the association of complex I into supercomplexes is required for the stabilization or even the biogenesis of this complex.
...
Die membranintegrierten, rotierenden F-Typ ATP-Synthasen zählen zu den essentiellen Komponenten der bakteriellen Energieversorgung. Ihre Rolle im zellulären Energiehaushalt bestehtin der Synthese von ATP unter Nutzung des transmembranen, elektrischen Ionengradienten (Mitchell 1961, Duncan et al. 1995, Noji et al. 1997, Kinosita et al. 1998). Die rotierenden ATP-Synthasen werden entsprechend der Kationenselektivität, die sie unter physiologischen Bedingungen zeigen, in zwei verschiedene Klassen eingeteilt, die H+-selektiven, sowiedie Na+-selektiven ATP-Synthasen. Hierbei bildet die Selektivität beider Klassen für einwertige Kationen (H+ oder Na+) eine essenzielle Grundlage für ihre Rolle im Energiehaushalt der bakteriellen Zellen. Jedoch gibt es nur eine begrenzte Anzahl von anaeroben Eubakterien und Archaeen, die noch einen auf Na+- Ionen basierenden Energiehaushalt besitzen. Gut charakterisierte Beispiele für Na+-selektive ATP-Synthasen bilden die F-Typ-Synthasen von I. tartaricus, P. modestum, sowie die V/A-Typ-Enzyme von E. hirae und A. woodii. Trotz der Unterschiede in der Kationenselektivitätder unterschiedlichen F-Typ ATP-Synthasen sind sie jedoch sowohl inihre Organisation, als auch hinsichtlich ihre Wirkungsweisen ähnlich. Das Ziel, der im Rahmen dieser Arbeit durchgeführten Forschung, bestand in der Identifizierung der Faktoren, die sowohl die hohen Selektivität, als auch die Affinität des in der Membran-eingebetteten Rotor-C-Rings der ATP-Synthasezu Protonen (H+) und Na+- Ionen beeinflussen. Die Untersuchungen wurden hierbei andem c11-Ring der F-Typ-ATP-Synthase aus dem anaeroben Bakterium Ilyobacter tartaricus durchgeführt, das hierbei als Modellsystem diente. Der untersuchte Ring zeigt unter physiologischen Bedingungen eine hohe Bindungsselektivität für Na+ Ionen, kann jedoch unter nicht-physiologischen Bedingungen auch Li+ und H+ Ionen binden und zur ATP-Synthese verwenden (Neumann et al. 1998).
Das Ziel, der im Rahmen dieser Arbeit durchgeführten Forschung, bestand in der Identifizierung der Faktoren, die sowohl die hohen Selektivität, als auch die Affinität des in der Membran-eingebetteten Rotor-C-Rings der ATP-Synthasezu Protonen (H+) und Na+- Ionen beeinflussen. Die Untersuchungen wurden hierbei andem c11-Ring der F-Typ-ATP-Synthase aus dem anaeroben Bakterium Ilyobacter tartaricus durchgeführt, das hierbei als Modellsystem diente. Der untersuchte Ring zeigt unter physiologischen Bedingungen eine hohe Bindungsselektivität für Na+ Ionen, kann jedoch unter nicht-physiologischen Bedingungen auch Li+ und H+ Ionen binden und zur ATP-Synthese verwenden (Neumann et al. 1998). Die Kd- und KM-Werte wurden verwendet, um die Na+ -Bindungsaffinität der C-Ringe bzw. ATP-Synthasen zu quantifizieren. Über die Selektivität wurdebeschrieben, welche Kationen an die C-Ringe und ATP-Synthasen binden können (z. B. H+/Na+/Li+, H+/Na+ - oder nur H+ Ionen).Das Verhältnis der absoluten Bindungsaffinitäten zwischen zwei Kationen (z. B. Kd (Na+)/Kd (H+)) wurde verwendet, um die Präferenz des Enzyms für eines der Ionen zu quantifizieren. Die Faktoren, dieder Kationenselektivität und der Affinität des I. tartaricus c-Rings zugrunde liegen, wurden mit Hilfe von Mutageneseexperimenten der Aminosäuren in der Ionenbindungsstelle untersucht. Im I. tartaricus-c-Ring erfolgt die Na+ Bindung an der Grenzfläche von zwei benachbarten c-Untereinheiten des c-Rings. An der Bindung der Na+-Ionen sind sowohl Aminosäuren aus Helix 1 (Gln32), sowie von Helix 2 (Val63, Ser66, Thr67 und Tyr70) beteiligt, die in der Nähe, des für den Mechanismusessentiellen Glu65 liegen. Insgesamt wurden 19 verschiedene, spezifische Einzel- und Doppelmutationen in die Sequenz des atpE-Gens eingeführt, die für die I. tarticus-ATP-Synthase-c-Untereinheit kodiert. Bei den Experimenten mit dem I. tartaricus c-Ring (Ser66, Thr67 und Tyr70) wurden drei polare Reste der Ionenbindungsstelle durch die polaren Reste (Ser67, Ile67 oder Leu67) oder hydrophobe Reste (Ala66, Gln67 und Phe70) ersetzt, während das geladene Glu65 durch die kürzere, aber immer noch geladene Seitenkette Asp65 ausgetauscht wurde. Zur Charakterisierung der monovalenten Kationenbindung durch die Wildtyp, sowie die mutierten C-Ringe von I.-tartaricus, wurde ein Ansatz verwendet, der biochemische (DCCD-Ionen-Kompetitionsassay) und biophysikalische (ITC) Methoden kombiniert.
Die Daten der in dieser Arbeit durchgeführten Experimente, zeigen, dass c-Ringe selektiv für H+ sind, solange in der Ionenbindungsstelle des c-Rings ein ionisierbarer Glu/Asp-Rest vorhanden ist. Die H+-Bindungsaffinität des c-Rings hängt von der Hydrophobizität der Reste ab, aus der die Ionenbindungsstelle aufgebaut ist.Jedoch ist die Zahl der Faktoren, die die Na+-Selektivität des C-Rings bestimmen, weitaus größer. Von den in dieser Arbeit untersuchten Faktoren war die Zahl der polaren Reste, die Wasserstoffbrücken zu Na+ bilden, die Co-Koordination von Na+ durch strukturell vorhandene Wassermoleküle und die Anwesenheit von negativ geladenen Resten besonders wichtig für die Bindung der Na+-Ionen an den Ring. Die hohe Bindungsaffinität des c-Rings für Na+-Ionen, wird sowohl durch Wechselwirkungen begünstigt die das gebundene Na+-Ion stabilisieren, als auch den gesamten atomaren Aufbau der Ionenbindestelle, der die enthalpiegetriebene Na+-Bindungan den c-Ring begünstigen. Im Rahmen dieser eingehenden Studien konnten zum ersten Mal die thermodynamischen Eigenschaften aufgeklärt werden, die der hohen Na+-Bindungsaffinität des c-Rings zugrunde liegen, sowie der Einfluss von Mutationen auf diese Parameter ermittelt werden. Durch zahlreiche Experimente mit ATP-Synthasen, die mit mutierten c-Ringen zusammengesetzt wurden, sollte eine Verbindung zwischen Veränderungen der H+- und der Na+-Bindungsaffinitäten und Unterschiede im Betrieb der ATP-Synthase aufgeklärt werden. Die wichtigste Schlussfolgerung, die sich aus dieser Arbeit ableiten lässt, ist, besteht darin, dass sich Na+/H+-selektiven ATP-Synthasen durch den Austausch von 1-2 Aminosäureresten innerhalb der rotierenden c-Ring-Ionenbindungsstelle in ausschließlich H+-selektive, vollfunktionelle ATP-Synthasen umwandeln lassen.
Im Rahmen dieser Arbeit wurden zum Vergleich die Strukturen der ATP-Synthasen von Arabidopsis thaliana, Asparagus officinalis, Allium cepa, Helianthus annus, Solanum tuberosum, Bos taurus und Saccharomyces cerevisiae gelöst. Die ATP-Synthase von S. cerevisiae konnte mit einer Auflösung von 19 Å gelöst werden. Der Winkel zwischen den zwei ATP-Synthase-Monomeren in dem ATP-Synthase-Dimer hatte für jede Spezies einen bestimmten Wert. Dieser Winkel änderte sich innerhalb einer Spezies nur wenig im Gegensatz zu Untersuchungen mit Einzelpartikelanalyse.
Die ATP-Synthase-Dimere aus den untersuchten Spezies besitzen unterschiedliche Winkel zwischen 78˚ und 122˚. Der Winkel des ATP-Synthase-Dimers aus S. tuberosum (122˚) viel größer als der in anderen Pflanzen (~98˚), B. taurus (105˚) und S. cerevisiae (78˚). Die Proben von S. tuberosum und B. taurus waren jedoch dünner, was den Winkel eventuell beeinflussen könnte. Um dies auszuschließen müssen in Zukunft weitere Untersuchungen durchgeführt werden.
Des Weiteren wurde im peripheren Stiel der ATP-Synthasen von allen Pflanzenspezies eine Dichte entdeckt, die in B. taurus und S. cerevisiae nicht vorhanden ist. Die Dichte könnte durch eine zusätzliche Untereinheit oder veränderte Untereinheit im Vergleich zu B. taurus und S. cerevisiae kommen.
Weiterhin wurde die Bildung von Reihen aus ATP-Synthase-Dimeren untersucht. Es wurden ATP-Synthase-Dimere von Polytomella sp. gereinigt und in Lipid rekonstituiert. Es wurde das ATP-Synthase-Dimer von Polytomella sp. verwendet, da dieses besonders stabil ist und während der Reinigung nicht zum ATP-Synthase-Monomer zerfällt. Zur Rekonstitution wurde die milde GRecon-Methode verwendet. Hierbei werden Membranproteine in einem Zuckergradienten gleichzeitig in Lipid rekonstituiert und nach ihrer Dichte getrennt. Abhängig von der Dichte der Proteoliposomen ist die Konzentration an Membranproteinen unterschiedlich. In Proteoliposomen mit einer hohen Konzentration bilden sich dünne Schichten in denen die ATP-Synthase-Dimeren Zickzack-Muster formen. Dies deutet darauf hin, dass das ATP-Synthase-Dimer die Membran verformt. In Proteoliposomen mit einer niedrigeren Konzentration an ATP-Synthase-Dimeren wurden runde Vesikel detektiert, in denen die ATP-Synthase-Dimere lange Reihen bilden und die Membran innerhalb jedes ATP-Synthase-Dimer ebenfalls verformt ist. Molekulare Simulationen bestätigen dieses Ergebnis.
Zudem wurde das ATP-Synthase-Dimer in zwei verschiedene Lipide ohne Cardiolipin rekonstituiert, da Cardiolipin ein Lipid ist welches in der bakteriellen und mitochondrialen Membran gefunden wurde und in hohen Konzentrationen in Membrankrümmungen lokalisiert ist (Huang et al., 2006), wie auch die ATP-Synthase-Dimere. Ohne Cardiolipin ist die Rekonstitution nicht geglückt beziehungsweise sind die ATP-Synthase-Dimere weniger gut zueinander angeordnet. Das deutet auf die Wichtigkeit von Cardiolipin in der Stabilisierung der Reihen von ATP-Synthase-Dimeren hin. Weitere Experimente mit verschiedenen ATP-Synthase-Dimeren in verschiedenen Lipiden sind nötig um dies zu untermauern.
Ein weiteres Ziel dieser Arbeit war es ein klonierbares Label zu etablieren, um ein bestimmtes Protein in Kryo-Elektronentomogramme zu identifizieren. Das Label sollte klein sein, um das zu identifizierbare Protein nicht zu beeinflussen und groß genug um in Kryo-Elektronentomogramme identifizierbar zu sein. In Einzelbildern wurde das 6 kDa große Metallothionein gebunden mit Gold identifiziert, wenn zwei Metallothioneine an dem gewünschten Protein kloniert wurden. Metallothionein besteht zu 33 % aus Cysteinen, welche Schwermetalle binden.
In meinen Studien habe ich bewiesen, dass drei Metallothioneine, gebunden mit Gold, in Kryo-Elektronentomogramme detektiert werden können. Jedoch tritt bei der Verwendung von Metallothionein durch die hohe Anzahl an Cysteinen vermehrt Aggregation auf. Bei meinen Untersuchungen fand ich heraus, dass auch das Maltose-Binde-Protein (MBP) ein Signal gleicher Intensität erzeugt. Durch Verwendung von MBP tritt aber keine Aggregation auf und man kann MBP auch zum Reinigen des Proteins verwenden.
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
During my thesis, I worked on two different membrane proteins. One is a bacterial secondary transporter and the second is a human mitochondrial calcium channel.
The first part of my thesis involves the structural and biochemical characterization of an L-carnitine/ γ-butyrobetaine antiporter from bacteria called CaiT. The aim of the project was to understand the Na+ independence of CaiT and to determine the crystal structures of CaiT in different conformations to expand the mechanistic understanding of substrate/ product antiport in CaiT.
The study revealed how a positively charged amino acid side chain (arginine 262) in CaiT could structurally and functionally mimic a sodium ion. Additionally, various crystal structures of CaiT obtained in this study demonstrate that the central substrate-binding site is highly dynamic and can accommodate the substrate in various orientations.
In the second part of my thesis, I was able to optimize the expression and purification conditions for the human mitochondrial calcium uniporter or the MCU. Understanding how this channel functions can help us unravel the mechanism of calcium uptake by mitochondria. Secondary structure prediction analysis in combination with mass spectrometry of degraded MCU products obtained during the purification of the full-length protein led to the identification of a stable MCU construct. This study resulted in the successful purification of milligram quantities of stable MCU protein for the first time. Further optimization may be required to obtain more homogenous protein that is amenable for crystallization.
ATP synthases are multi-subunit membrane enzymes, which utilize the energy stored in a transmembrane electrochemical ion gradient to produce adenosine-5´-triphosphate (ATP), the universal energy carrier in biological systems. Research on these important enzymes goes back more than 50 years and has produced innumerable studies. The F-type ATP synthase consists of two functionally distinct, but tightly coupled subcomplexes, the water-soluble F1 and the membrane-embedded Fo complex. In its simplest form, F1 consists of five different subunits with a stoichiometry of α 3β3γδε, and harbors three catalytic centers in the α 3β3-headpiece, while Fo consists of three different subunits in a stoichiometry of ab2cn, where n varies between 8 to 15 depending on the species. From a mechanistic standpoint, the complex can also be divided into two different units, namely a stator, α3β3δ-ab2, and a rotor, γε-cn. The enzyme utilizes the energy stored in a transmembrane electrochemical gradient of protons, or in some cases Na+, to drive ATP synthesis. In particular, the downhill translocation of these ions across the Fo complex drives rotation of the γε-cn unit, which is then transduced to the active centers, catalyzing the phosphorylation of adenosine-5`-diphosphate (ADP) with inorganic phosphate (Pi), and the release of ATP....
Plants absorb sunlight via photosynthetic pigments and convert light energy intochemical energy in the process of photosynthesis. These pigments are mainly bound to antenna protein complexes that funnel the excitation energy to the photosynthetic reaction centres. The peripheral antenna of plant photosystem II (PSII) consists of the major light-harvesting complex of PSII (LHC-II) and the minor LHCs CP29, CP26 and CP24. Light intensity can change frequently and plants need to adapt to high-light conditions in order to avoid photodamage. When more photons are absorbed than can be utilised by the photosynthetic machinery, excessive excitation energy is dissipated as heat by short-term adaptation processes collectively known as non-photochemical quenching (NPQ). A decrease in PSII antenna chlorophyll (Chl) fluorescence yield and a reduction in the average Chl fluorescence lifetime are associated with NPQ. The main component of NPQ is the so-called energy-dependent quenching (qE), and it is triggered by the rapid drop in thylakoid lumenal pH resulting from the plant’s photosynthetic activity. This process is thought to take place at the PSII antenna complexes, which therefore not only capture and transfer light energy but are also involved in balancing the energy flow. The decrease in lumenal pH acivates the enzyme violaxanthin de-epoxidase (VDE), which converts the xanthophyll violaxanthin (Vio) into zeaxanthin (Zea) in the xanthophyll cycle. In addition, the PSII subunit PsbS was discovered to be essential for qE by screening qE-deficient Arabidopsis thaliana mutants. This membrane protein is considered a member of the LHC superfamily, which also includes LHC-II and the minor LHCs. Previous studies on PsbS isolated either from native source or refolded in vitro have produced inconsistent results on its pigment binding capacity. Interestingly, a pH-dependent change in the quaternary structure of PsbS under high light conditions has been reported. This observed dimer-tomonomer transition very likely follows the protonation of lumenal glutamates upon the drop in pH and is accompanied by a change in PSII supercomplex localisation. PsbS dimers are preferentially found in association with the PSII core, whereas PsbS monomers co-localise with LHC-II.Despite the identification of !pH, Zea and PsbS as key players in qE, both the nature of the quencher(s) as well as the underlying molecular mechanism leading to excess energy dissipation still remain unknown. Several models have been put forward to explain the reversible switch in the antenna from an energy-transmitting to a quenched state. Proposals include a simple pigment exchange of Vio for Zea, and aggregation or an internal conformational change of LHC-II. Charge transfer (CT)quenching in the minor LHCs or quenching by carotenoid dark state (Car S1)-Chl interactions have also been suggested. However, none of these qE models has so far been capable of accommodating all the physiological observations and available experimental data. Most importantly, the function of PsbS remains an enigma. A recent qE model suggested that monomerisation of PsbS enables the protein to transiently bind a carotenoid and form a quenching unit with a Chl of a PSII LHC. In view of the various proposed qE mechanisms, this thesis aimed at understanding the interplay of the different qE components and the contribution of the PSII subunits LHC-II, the minor LHCs and PsbS to qE. The initial approach was to investigate the properties of the PSII subunits in the most simple in vitro model system, namely in detergent solution. For this purpose, LHC-II was isolated either from native source or refolded from recombinantly produced protein. Investigation of the minor LHCs and PsbS required heterologous expression and refolding. In addition, experiments were performed on aggregated LHC-II. Aggregates of LHC-II have been used as a popular model system for qE because they exhibit highly quenched Chl fluorescence. At the final stage of this doctoral work, a more sophisticated model system to approximate the thylakoid membrane was developed by reconstitution of the PSII subunits LHC-II and PsbS into liposomes. This system not only allowed for investigation of these membrane proteins in their native environment, but also for mimicking the xanthophyll cycle by distribution of Zea within the membrane as well as !pH by outside buffer exchange. The role of Zea in qE was first investigated with detergent solubilised antenna proteins. The requirement of this xanthophyll for qE is well-known, but the specific contribution to the molecular quenching mechansim is unclear. Previous work had shown that replacement of Vio for Zea in LHC-II was not sufficient to induce Chl fluorescence quenching in Zea-LHC-II, as suggested by the so-called molecular gearshift mechanism. However, by means of selective two-photon excitation spectroscopy, an increase in electronic interactions between Car S1 and Chls was observed for LHC-II upon lowering the pH of the detergent buffer. Electronic Car S1-Chl coupling became even stronger when Zea-LHC-II was probed. The extent of Car S1-Chl coupling correlated directly with the extent of Chl fluorescence quenching, in a similar way as observed previously in live plants under high-light conditions. However, very similar results were obtained with LHC-II aggregates. This implied that the increase in electronic interactions and fluorescence quenching was independent of Zea and low pH. Further experiments on aggregates of LHC-II Chl mutants indicated that the targeted pigments were also not essential for the observed effects. It is proposed that the same molecular mechanism causes an increase in electronic Car S1-Chl interactions and Chl fluorescence quenching in Zea-LHC-II at low pH as well as in aggregated LHC-II. Most likely, surface exposed pigments form random quenching centres in both cases. On the other hand, it was possible that Zea could act as a direct quencher of excess excitation energy in the minor LHCs. However, enrichment of refolded CP29, CP26 and CP24 with Zea did not lead to a change in the Chl excited state lifetime. Formation of a carotenoid radical cation, previously implied in CT quenching, was also not observed, although artificial generation of such a radical cation was principally possible as shown for CP29. During the course of this work, a study reporting the formation of Zea radical cations in minor LHCs was published. Therefore, Zea-enriched minor LHCs were again investigated on the experimental apparatus used in the reported study. Indeed, the presence of at least one carotenoid radical cation for each minor complex was detected. It is suggested that either the preparation method of incubating the refolded minor LHCs with Zea in contrast to refolding the complexes with only Zea and lutein causes the observed differences or that the observed spectral radical cation signatures are due to experimental artifacts. While the experiments with LHC-II and the minor LHCs gave useful insights into the putative qE mechanism, the quencher site and the mode of action of Zea could still not be unambiguously identified. Most importantly, these studies could not explain the function of the qE keyplayer PsbS. Therefore, the focus of the work was shifted to PsbS protein production, purification and characterisation. In view of inconsistent reports on the pigment binding capacity of this PSII subunit, refolding trials with and without photosynthetic pigments were conducted. The formation of a specific pigmentprotein complex typical for other LHCs was not observed and neither was the earlier reported “activation” of Zea for qE by binding to this protein. Nevertheless, PsbS refolded without pigments displayed secondary structure content in agreement with previous studies, indicating pigment-independent folding. Reconstitution of pigmentfree, refolded PsbS into liposomes confirmed that the protein is stable in the absence of pigments. Zea distributed in PsbS-containing liposomes also showed no spectral alteration that would indicate its “activation”. With the ability to reconstitute PsbS, it was then possible to proceed to modelling qE in a proteoliposome system. For this purpose, PsbS was co-reconstituted with LHC-II, which has been reported to interact with PsbS. One-photon excitation (OPE) and two-photon excitation (TPE) spectroscopy measurements were performed on LHC-II- and LHC-II/PsbS-containing liposomes. This enabled both quantification of Chl fluorescence quenching as well as determination of the extent of electronic Car S1-Chl interactions. The effect of Zea was investigated by incorporating it in the proteoliposome membrane. It was shown that Zea alone was not able to induce significant Chl fluorescence quenching when only LHC-II was present. However, when LHC-II and PsbS were co-reconstituted, pronounced Chl fluorescence quenching and an increase in electronic Car S1-Chl interactions were observed and both effects were enhanced when Zea was present. Western blot analysis indicated the presence of a LHC-II/PsbS-heterodimer in these proteoliposomes. In addition to the OPE and TPE measurements, the average Chl fluorescence lifetime was determined in detergent-free buffer at neutral pH and directly after buffer exchange to low pH. No significant changes in the average lifetime were observed for LHC-II proteoliposomes when either Zea was present or after exchange for low pH buffer. This indicated that Zea alone cannot act as a direct quencher, which concurs with the OPE measurements. Moreover, the complex was also properly reconstituted as no aggregation or significant Chl fluorescence quenching were observed. The average lifetime was not significantly affected in LHC-II/PsbS-proteoliposomes, independent of Zea or pH. However, a shortlived component in the presence of a long-lived component was not resolvable with the time resolution of the fluorescence lifetime apparatus.
Implications for qE model systems and the in vivo quenching mechanism are discussed based on the experiments in detergent solution, on LHC-II aggregates and with the proteoliposome model system.