Open Access

Sandra Johanna Ullrich

• In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa. First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi. A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise. For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution. The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles. Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95. Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
• In der hier vorgelegten Dissertation wird das integrale Membranprotein Diacylglycerin-Kinase (DAGK) aus E.coli mit Festkörper-Nuklearmagnetischer Resonanz (FK-NMR) Spektroskopie untersucht. FK-NMR ist eine etablierte Methode für Hypothesen-getriebene Studien an Membranproteinen zur Aufklärung ihrer Funktionsmechanismen und gewinnt ebenfalls immer größere Bedeutung in der Strukturbiologie[109,110,108,128,129,180]. Die besondere Stärke dieser Methode liegt in der Möglichkeit, Membranproteine direkt in der Lipiddoppelschicht zu studieren. Das Ziel dieser Dissertation ist es, durch Integration von Daten über Enzymkinetik, Proteinstruktur und Proteinrückgratdynamik Ein-blicke in dieses Enzym zu gewinnen. Die dabei entwickelten Methoden und Techniken bilden eine wesentliche Voraussetzung zur Aufklärung der Struktur-Funktions-Zusammenhänge von Membran-proteinen im Allgemeinen und der molekularen Grundlagen des Lipid Signaling im Besonderen. DAGK repräsentiert eine Familie der prokaryotischen Kinasen, welche eine wichtige Rolle in der mikrobiellen Physiologie spielen[31,32,33,34]. Die biologische Funktion von DAGK ist der Transfer der γ-Phosphatgruppe von Mg*ATP aus dem Cytoplasma auf Diacylglycerol (DAG) in der Membran zur Bildung von Phosphatidylsäure (PA)[6]. Damit partizipiert DAGK im Membran-Oligosaccharid-Weg, welcher Reaktionen auf beiden Seiten der Plasmamembran involviert[31,34]. Bemerkenswert ist, dass DAGK weder Struktur- noch Sequenzähnlichkeiten zu anderen Kinasen aufweist[12]. Typische Sequenzmotive anderer Enzyme, die Phosphoryltransferreaktionen katalysieren, liegen nicht vor[13]. In seiner physiologischen Form bildet DAGK ein Homotrimer, mit neun Transmembranhelices und drei katalytisch aktiven Zentren, welche zusammen ein Molekulargewicht von 39,6 kDa ergeben[65]. Jedes Monomer weist drei Transmembranhelices sowie zwei kurze amphiphatische Helices am N-Terminus auf[132]. Van Horn und Kim bestimmten 2009 die 3D Proteinrückgratstruktur von DAGK in Detergenzmicellen mittels Lösungsmittel-NMR[63]. Dies stellte auf Grund der Größe und Komplexität des Proteins einen Durchbruch in der Strukturbiologie mit Lösungsmittel-NMR dar. In dieser Struktur ist die dritte Transmembranhelix eines jeden Monomers zwischen die erste und zweite Transmembranhelix des links angrenzenden Monomers eingelagert. Dabei wird die aktive Tasche durch eine tunnelartige Struktur der Transmembranhelices gebildet, welche von einem Loop zwischen Transmembranhelix 2 und 3 überdacht wird[63]. Die grüne Variante des Proteorhodopsin (GPR) ist das zweite Protein, welches in der vorliegenden Arbeit auf Grund seiner Modelleigenschaften verwendet wurde. Ursprünglich wurde GPR aus halophilen γ-Proteobakterien isoliert, die in großen Mengen in Ozeanen vorkommen[74]. GPR ist ein integrales Membranprotein mit sieben Transmembranhelices und einer Größe von 27 kDa. Als lichtgetriebene Protonenpumpe wandelt GPR Lichtenergie in elektrochemisches Potential durch Transport von Protonen über eine Membran um[80]. Evolutionär bedingte Adaptation der umgebungsbedingten Absorptionseingeschaften erzeugte verschiedene Varianten des Proteorhodopsin[79]. Allen Varianten ist gemeinsam, dass die Absorption von Lichtquanten am kovalent gebundenen Chromophor Retinal eine Konformationsänderung im Protein induziert. Das Protein transportiert beim Durchlaufen eines mehrstufigen Photozyklus, bestehend aus metastabilen Intermediaten, Protonen[80]. Bei diesem Prozeß kann momentan eine Signaling-Funktion nicht ausgeschlossen werden[86]. Im Rahmen dieser Arbeit wurden verschiedene Methoden zur Probenpräparation verwendet: Molekularbiologische Methoden zur Herstellung der Vektoren für die Expression der jeweiligen Proteine oder ihrer Mutanten in vivo und in vitro[150] sowie Methoden für die nachfolgende Isolation, Reinigung und Rekonstitution in synthetische Lipidmembranen. Homogenität und biologische Aktivität des Proteins unter den gewählten spektroskopischen Bedingungen sind von essentieller Bedeutung für funktionale Studien. Überprüft werden können diese zum Beispiel in einem biochemischen Assay ...