TY - THES A1 - Hiller, Matthias T1 - Sample preparation of membrane proteins suitable for solid-state MAS NMR and development of assignment strategies T1 - Präparation von Membranproteinen für Strukturuntersuchungen mittels Festkörper MAS NMR und die Entwicklung von Zuordnungsstrategien N2 - Although the basic structure of biological membranes is provided by the lipid bilayer, most of the specific functions are carried out by membrane proteins (MPs) such as channels, ion-pumps and receptors. Additionally, it is known, that mutations in MPs are directly or indirectly involved in many diseases. Thus, structure determination of MPs is of major interest not only in structural biology but also in pharmacology, especially for drug development. Advances in structural biology of membrane proteins (MPs) have been strongly supported by the success of three leading techniques: X-ray crystallography, electron microscopy and solution NMR spectroscopy. However, X-ray crystallography and electron microscopy, require highly diffracting 3D or 2D crystals, respectively. Today, structure determination of non-crystalline solid protein preparations has been made possible through rapid progress of solid-state MAS NMR methodology for biological systems. Castellani et. al. solved and refined the first structure of a microcrystalline protein using only solid-state MAS NMR spectroscopy. These successful application open up perspectives to access systems that are difficult to crystallise or that form large heterogeneous complexes and insoluble aggregates, for example ligands bound to a MP-receptor, protein fibrils and heterogeneous proteins aggregates. Solid-state MAS NMR spectroscopy is in principle well suited to study MP at atomic resolution. In this thesis, different types of MP preparations were tested for their suitability to be studied by solid-state MAS NMR. Proteoliposomes, poorly diffracting 2D crystals and a PEG precipitate of the outer membrane protein G (OmpG) were prepared as a model system for large MPs. Results from this work, combined with data found in the literature, show that highly diffracting crystalline material is not a prerequirement for structural analysis of MPs by solid-state MAS NMR. Instead, it is possible to use non-diffracting 3D crystals, MP precipitates, poorly diffracting 2D crystals and proteoliposomes. For the latter two types of preparations, the MP is reconstituted into a lipid bilayer, which thus allows the structural investigation in a quasi-native environment. In addition, to prepare a MP sample for solid-state MAS NMR it is possible to use screening methods, that are well established for 3D and 2D crystallisation of MPs. Hopefully, these findings will open a fourth method for structural investigation of MP. The prerequisite for structural studies by NMR in general, and the most time consuming step, is always the assignment of resonances to specific nuclei within the protein. Since the last few years an ever-increasing number of assignments from solid-state MAS NMR of uniformly carbon and nitrogen labelled samples is being reported, mostly for small proteins of up to around 150 amino acids in length. However, the complexity of the spectra increases with increasing molecular weight of the protein. Thus the conventional assignment strategies developed for small proteins do not yield a sufficiently high degree of assignment for the large MP OmpG (281 amino acids). Therefore, a new assignment strategy to find starting points for large MPs was devised. The assignment procedure is based on a sample with [2,3-13C, 15N]-labelled Tyr and Phe and uniformly labelled alanine and glycine. This labelling pattern reduces the spectral overlap as well as the number of assignment possibilities. In order to extend the assignment, four other specifically labelled OmpG samples were used. The assignment procedure starts with the identification of the spin systems of each labelled amino acid using 2D 13C-13C and 3D NCACX correlation experiments. In a second step, 2D and 3D NCOCX type experiments are used for the sequential assignment of the observed resonances to specific nuclei in the OmpG amino acid sequence. Additionally, it was shown in this work, that biosynthetically site directed labelled samples, which are normally used to observe long-range correlations, were helpful to confirm the assignment. Another approach to find assignment starting points in large protein systems, is the use of spectroscopic filtering techniques. A filtering block that selects methyl resonances was used to find further assignment starting points for OmpG. Combining all these techniques, it was possible to assign nearly 50 % of the observed signals to the OmpG sequence. Using this information, a prediction of the secondary structure elements of OmpG was possible. Most of the calculated motifs were in good aggreement with the crystal structures of OmpG. The approaches presented here should be applicable to a wide variety of MPs and MP-complexes and should thus open a new avenue for the structural biology of MPs. N2 - Biologische Membranen bestehen hauptsächlich aus Lipiden, ihre Funktion wird jedoch vor allem durch die eingebetteten Membranproteine (z.B. Kanäle, Ionenpumpen und Rezeptoren) bestimmt. Mutationen in dieser Proteinklasse können zum Auftreten verschiedener Krankheitsbilder führen, weshalb die Untersuchung der dreidimensionalen Struktur von Membranproteinen nicht nur von strukturbiologischem, sondern auch von pharmakologischem Interesse ist. In den letzten Jahren wurde eine Methode, die Festkörper NMR Spektroskopie, für Strukturuntersuchungen an Proteinproben im festen Aggregatzustand entwickelt. Diese Arbeit beschäftigt sich mit drei verschiedenen Präparationsarten von Membranproteinen, die eine Aufnahme von hochaufgelösten Festkörper NMR Spektren erlauben. Als Modelsystem wurde das Protein G der äußeren Membrane (outer membrane protein G, OmpG) von Escherichia coli gewählt. Eine wichtige Vorraussetzung zur Berechnung der Proteinstruktur aus den NMR-Spektren, ist die Zuordnung der einzelnen Signale zur jeweiligen Aminosäure in der Proteinsequenz. In dieser Arbeit wurde eine Methode entwickelt, die das Auffinden von Startpunkten für die sequentielle Zuordnung in großen Membranproteinen, wie zum Bsp. OmpG (281 Aminosäuren), erlaubt. Multidimensionale NMR Experimente mit verschieden spezifisch markierten Proben wurden durchgeführt und ermöglichten die Zuordnung von 50 % der NMR Signale der OmpG Proteinsequenz. Zur Überprüfung der gewonnenen Daten wurden diese zur Vorhersage von Sekundärstrukturelementen genutzt. Es konnte gezeigt werden, dass die berechneten Strukturmotive in guter Übereinstimmung zu den bisher veröffentlichten OmpG Strukturen liegen. Die in dieser Arbeit angewendeten Methoden sollten auf eine Vielzahl anderer Membranprotein anwendbar und somit einen neuen Weg zur Strukturbiologischen Untersuchung von Membranproteinen eröffnen. KW - Membranproteine KW - Festkörper NMR Spektroskopie KW - Proteinstruktur KW - OmpG KW - Membrane protein KW - solid-state MAS NMR KW - protein structure KW - OmpG Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-37246 ER - TY - THES A1 - Dolniak, Blazej T1 - Functional characterisation of NIC2, a member of the MATE family from Arabidopsis thaliana (L.) Heynh. T1 - Funktionale Charakterisierung von NIC2, einem Mitglied der MATE Familie aus Arabidopsis thaliana (L.) Heynh. N2 - The multidrug and toxic compounds extrusion (MATE) family includes hundreds of functionally uncharacterised proteins from bacteria and all eukaryotic kingdoms except the animal kingdom, that function as drug/toxin::Na+ or H+ antiporters. In Arabidopsis thaliana the MATE family comprises 56 members, one of which is NIC2 (Novel Ion Carrier 2). Using heterologous expression systems including Escherichia coli and Saccharomyces cerevisiae, and the homologous expression system of Arabidopsis thaliana, the functional characterisation of NIC2 was performed. It has been demonstrated that NIC2 confers resistance of E. coli towards the chemically diverse compounds such as tetraethylammonium chloride (TEACl), tetramethylammonium chloride (TMACl) and a toxic analogue of indole-3-acetic acid, 5-fluoro-indole-acetic acid (F-IAA). Therefore, NIC2 may be able to transport a broad range of drug and toxic compounds. In wild-type yeast the expression of NIC2 increased the tolerance towards lithium and sodium, but not towards potassium and calcium. In A. thaliana, the overexpression of NIC2 led to strong phenotypic changes. Under normal growth condtions overexpression caused an extremely bushy phenotype with no apical dominance but an enhanced number of lateral flowering shoots. The amount of rossette leaves and flowers with accompanying siliques were also much higher than in wild-type plants and the senescence occurred earlier in the transgenic plants. In contrast, RNA interference (RNAi) used to silence NIC2 expression, induced early flower stalk development and flowering compared with wild-type plants. In additon, the main flower stalks were not able to grow vertically, but instead had a strong tendency to bend towards the ground. While NIC2 RNAi seedlings produced many lateral roots outgrowing from the primary root and the root-shoot junction, NIC2 overexpression seedlings displayed longer primary roots that were characterised by a 2 to 4 h delay in the gravitropic response. In addition, these lines exhibited an enhanced resistance to exogenously applied auxins, i.e. indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) when compared with the wild-type roots. Based on these results, it is suggested that the NIC2 overexpression and NIC2 RNAi phenotypes were due to decreased or increased levels of auxin, respectively. The ProNIC2:GUS fusion gene revealed that NIC2 is expressed in the stele of the elongation zone, in the lateral root cap, in new lateral root primordia, and in pericycle cells of the root system. In the vascular tissue of rosette leaves and inflorescence stems, the expression was observed in the xylem parenchyma cells, while in siliques it was also in vascular tissue, but as well in the dehiscence and abscission zones. The organ- and tissue-specific expression sites of NIC2 correlate with the sites of auxin action in mature Arabidopsis plants. Further experiments using ProNIC2:GUS indicated that NIC2 is an auxin-inducible gene. Additionally, during the gravitropic response when an endogenous auxin gradient across the root tip forms, the GUS activity pattern of the ProNIC2:GUS fusion gene markedly changed at the upper side of the root tip, while at the lower side stayed unchanged. Finally, at the subcellular level NIC2-GFP fusion protein localised in the peroxisomes of Nicotana tabacum BY2 protoplasts. Considering the experimental results, it is proposed that the hypothetical function of NIC2 is the efflux transport which takes part in the auxin homeostasis in plant tissues probably by removing auxin conjugates from the cytoplasm into peroxisomes. N2 - "Multidrug and Toxic Compounds Extrusion" (MATE) – Proteine sind Membranproteine, die eine Vielzahl komplexer und giftiger Substanzen transportieren können. Sie sind weit verbreitet und kommen in Bakterien und Höheren Organismen mit Ausnahme des Tierreichs vor. Insgesamt gibt es hunderte von bisher kaum untersuchten Genen dieser Familie, die eine hohe Sequenzhomologie aufweisen. In der Pflanze Arabidopsis thaliana wurden 56 Gene der MATE - Familie zugeordnet. Eines von ihnen, der "Novel Ion Carrier 2" (NIC2) wurde näher charakterisiert. Dafür wurden heterologe Expressionssysteme wie Bakterien (Escherichia coli) und Hefe (Saccharomyces cerevisiae) genutzt und transgene Pflanzen (Arabidopsis thaliana) hergestellt. Es wurde gezeigt, dass NIC2 Bakterien eine Resistenz gegenüber mehreren giftigen Stoffen verlieh. In Hefe erhöhte NIC2 die Salztoleranz gegenüber Lithium und Natrium, aber nicht gegenüber Kalium und Kalzium. Das deutet darauf hin, dass NIC2 diese Stoffe transportieren kann und so zur Entgiftung beziehungsweise erhöhter Stresstoleranz beiträgt. In Pflanzen führte die Überexpression von NIC2 zu dramatischen Änderungen im Wachstum. Die Pflanzen waren buschig ohne zentralen Blütenstand, hatten jedoch eine höhere Anzahl von Blättern und Blüten und längere Wurzeln mit einer im Vergleich zu den Wildtyppflanzen verzögerten gravitropen Antwort. In Gegensatz dazu entwickelten Pflanzen, in denen die Expression von NIC2 gehemmt wurde, früh einen zentralen Blütenstand, der allerdings nicht gerade wuchs, sondern die Tendenz hatte, sich zum Boden zu biegen. Das Wurzelsystem bestand aus einer Hauptwurzel und vielen sekundären Wurzeln und war im Vergleich zu den Wildtyppflanzen besser entwickelt. Vermutlich kann die Wuchsform auf einen veränderten Gehalt des Pflanzenhormons Auxin zurückgeführt werden. Die Expression von NIC2 wird durch Auxin induziert. Experimente, in denen die Aktivität eines Gens mit Hilfe eines Reportergens nachgewiesen wird, zeigten, dass NIC2 in Wurzeln, Blättern, Blütenstielen, Blüten und Schoten aktiv ist. Innerhalb der Zelle ist NIC2 in Peroxisomen lokalisiert. Peroxisomen sind kleine Organellen, die eine Rolle im Hormonstoffwechsel spielen können, wie z.B. im Fall von Auxinen. Die Daten sprechen dafür, dass NIC2 eine Funktion beim Auxintransport und somit bei der Auxin-Homöostase hat. KW - Ackerschmalwand KW - Auxine KW - Membranproteine KW - Arabidopsis KW - membrane protein KW - auxin Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-5372 ER -