- Biochemie und Chemie (16) (remove)
- Insights into a Lipid Regulator by Solid-State MAS NMR: Kinetic and Structure-Functional Studies on Diacylglycerol Kinase (2013)
- 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) as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found. 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. 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. 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. 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, 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 was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs, 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, 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. 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. 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.
- Biophysical Studies of Lipid Membranes by Solid State NMR and Molecular Dynamics Simulations (2013)
- Biological membranes separate the cell interior from the outside and have diverse functions from signal transduction, apoptosis to transportations of ions and small molecules in and out of the cell. Most of these functions are fulfilled by proteins incorporated in the membrane. However, lipids as the main component of membrane not only serve as structural element for bilayer formation but they are also directly involved e.g. signalling processes and bilayer properties are important to mediate protein interactions. To fully understand the role of lipids, it is necessary to develop a molecular understanding of how certain membrane components modify bulk bilayer structure and dynamics. Membranes are known to have many different motions in different conditions and time scales. Temperature, pH, water content and many other conditions change membrane dynamics in a high degree. In addition to this, time scales of motions in membranes vary from ns to ms range corresponding to fast motion and slow motion, respectively. Therefore, membranes are needed to be studied systematically by varying the conditions and using methods to investigate motions in various time scales separately. The aim of this study was therefore perform a combined solid-state NMR / molecular dynamics study on model membranes. Different substrates, such as potential drugs, polarizing agents and signaling lipids were incorporated into bilayers and their location within the membrane and their effect onto the membrane was probed. NSAIDs (non-steroidal anti-inflammatory drugs), pirinixic acid derivatives, ceramides and polarizing agents were the substrates for membranes in this study. There were several experimental methods that were applied in order to investigate effects of these substrates on membrane dynamics. Different kind of phospholipids including POPC, DMPC and DPPC were used. In addition to experimental work, with the information gathered from solid state NMR experiments molecular dynamics simulations were performed to obtain more information about the membranes at the molecular level. As a result, combination of solid-state NMR with molecular dynamics simulations provides very systematic way of investigating membrane dynamics in a large range of time scales. Pirinixic acid derivatives were special interest of this study because of their activity on peroxisome proliferator-activated receptor (PPAR) as an agonist as well as on enzymes of microsomal prostaglandin E2 synthase-1 (PGE2s) -1 and 5-lipoxygenase (5-LO) as dual inhibitor. Two potent pirinixic acid derivatives, 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoic acid (compound 2) and 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoate (compound 3), have been worked and their insertion depts were investigated by combining of solid state NMR and molecular dynamics simulations. Both experimental and theoretical results pointed out that compound 3 was inserted the phospholipid bilayer more deeply than 2. NSAIDs – lipid mixtures have been also studied here. It is known that consumption of NSAIDs as in mixture with lipids results much fewer side effects than consumption of the drugs alone. Thus, it is crucial to understand interactions of NSAIDs with lipids and investigate the possible complex formation of drugs with lipids. In this study, interactions of three widely used NSAIDs, ibuprofen, diclofenac and piroxicam, with DPPC were investigated by solid-state NMR. 1H and 31P NMR results depicted that ibuprofen and diclofenac had interactions with lipids, which is an indication of drug-lipid complex formation whereas piroxicam didn’t show any interactions with lipids suggesting that no complex formation occurred in the case of piroxicam. Ceramides are known to play key roles in many cell processes and many studies showed that the functions of ceramides are related with the ceramide effects on biological membranes. Therefore, in this study, influences of ceramides on biophysics of lipid bilayers were investigated by using various solid state NMR techniques and molecular dynamics simulations. Results from molecular dynamics simulations clearly showed that ceramide and lipids have strong interactions. More evidences about ceramide-lipid interactions were provided from 1H and 14N NMR results. In addition, it was indicated by both simulation and experimental methods that ceramide increased the rigidity of DMPC by increasing chain order parameters. BTbk is a biradical, which is used as polarizing agent for dynamic nuclear polarization (DNP) experiments and found to be more efficient than other widely used polarizing agents such as TOTAPOL. Since it is a hydrophobic compound, which prefers to stay inside lipid bilayer it is important to investigate the location and orientation of bTbk along the bilayer in order to understand its enhancement profile in DNP measurements. In this study, both NMR relaxation time measurements and molecular dynamics simulations revealed that bTbk tends to stay more close to hydrophobic chain of lipids than the interfacial part of lipids at bilayer surface. In the first part of this work, a brief introduction on lipid membranes as well as a theoretical summary on both methods of solid-state NMR and molecular dynamics simulations is given. Then, in the second part methodology is introduced for both solid-state NMR spectrometer and theoretical calculations. Afterwards, results of different membrane systems are discussed in the following parts for both solid state NMR and MD. Finally, in the last part, a summary and the conclusion of the overall results together with some future plans are explained.
- Solid-state NMR investigations of the ATP binding cassette multidrug transporter LmrA (2006)
- The development of resistance to multiple drugs is a major problem in treatment of number of infectious diseases and cancer. The phenomenon of multidrug resistance (MDR) is based on the synergetic interplay of a number of mechanisms such as target inactivation, target alteration, prevention of drug influx as well as active extrusion of drugs from the cell. The latter is mediated by over-expression of multidrug efflux pumps. The first discovered and the best characterized until now the human MDR transporter is P-glycoprotein. It is a member of the ATP binding cassette (ABC) superfamily and acts as an active transporter for a variety of anticancer agents using the energy released by ATP hydrolysis. The closest structure and functional homologue of P-glycoprotein found in bacteria is LmrA from Lactococcus lactis. The major goals of this work are to establish the selective isotope labelling of LmrA in Lactococcus lactis, to optimize LmrA sample preparation for solid-state NMR, and finally to perform first solidstate NMR investigations on LmrA shedding light on its catalytic cycle and substrate binding. For a long time the solid-state NMR applications to biological science has been limited to investigation of small molecules mostly. Recently, the solid-state NMR methods have shown potential for structuraland non-perturbing, site directed functional studies of large membrane proteins as well as ligands bound to them. However, to our knowledge neither selective isotope amino acid labelling of any ABC transporter, nor NMR investigations on full-length ABC transporter have been reported to date. Solidstate NMR experiments on a membrane protein require reconstitution of purified proteins into a membrane environment at a high density and either isotopic enrichment of the protein or bound drugs or inhibitors. Therefore, the large quantities of LmrA reconstituted at a high density in lipid membranes, sufficient for advanced NMR studies have been produced and its functional state in reconstituted form has been assessed. In the next step, a procedure for cost effective selective amino acids isotope labelling of LmrA in Lactococcus lactis has been established. Using this protocol deuterium alanine labelled LmrA reconstituted into E. coli liposomes has been prepared. Deuterium NMR has been used extensively to assess the proteins dynamics in past. However, it has never been applied to ABC transporter. Here, we report 2H NMR on selective alanine isotope labelled LmrA which has been used to shed light on the dynamics changes in the protein occurred under AMP-PNP, non-hydrolysable ATP analogue, binding and in ATP/ADP-Vanadate trapped state. It has been found that the major conformation changes affecting the protein motional characteristics occur in the ATP binding domains but not in the transmembrane domains. Additionally, the binding of several substrates to LmrA has been studied by fluorescence spectroscopy as well as by 19F and 31P solid-state NMR. The binding constants for several LmrA substrates have been obtained by fitting the concentration dependant tryptophan intrinsic fluorescence quenching curves. Based on the fluorescence studies and solid-state NMR data, the conformation changes in LmrA under substrate binding have been discussed. In addition, the preferable location of nine LmrA and P-glycoprotein substrates within the model membrane has been studied via 1H-MAS-NOESY-NMR. The results have been interpreted with respect to LmrA and P-glycoprotein binding site accessibility from the membrane interface region.
- NMR, EM and functional studies on TBsmr, a small multidrug transporter from M. tuberculosis (2008)
- Antibiotic resistance of pathogenic bacteria is a major worldwide problem. Bacteria can resist antibiotics by active efflux due to multidrug efflux pumps. The focus of this study has been the mycobacterial multidrug transporter TBsmr because it belongs to the small multidrug resistance (SMR) family whose members are a paradigm to study multidrug efflux due to their small size. SMR proteins are typically 11-12 kDa in size and have a four-transmembrane helix topology. They bind cationic, lipophilic antibiotics such as ethidium bromide (EtBr) and TPP+, and transport them across the membrane in exchange for protons. To understand the molecular mechanism of multidrug resistance, we have to gain information about the structure and function of these proteins. The research described in this thesis aimed to deduce details about the topology, transport cycle and key residues of TBsmr using biophysical techniques. Solid-state NMR (ssNMR) can provide detailed insight into structural organization and dynamical properties of these systems. However, a major bottleneck is the preparation of mg amounts of isotope labeled protein. In case of proteoliposomes, the problem is compounded by the presence of lipids which have to fit into the small active volume of the ssNMR rotor. In Chapter 3, an enhanced protein preparation is described which yields large amounts of TBsmr reconstituted in a native lipid environment suitable for further functional and structual studies. The achieved high protein-to-lipid ratios made a further characterization by ssNMR feasible. The transport activity and oligomeric state of the reconstituted protein in different types of lipid was studied as shown in Chapter 4. The exact oligomeric state of native SMR proteins is still uncertain but a number of biochemical and biophysical studies in detergent suggest that the minimal functional unit capable of binding substrate is a dimer. However, binding assays are not ideal since a protein may bind substrate without completing the transport cycle which can only be shown for reconstituted protein in transport assays.By combining functional data of a TPP+ transport assay with information about theoligomeric state of reconstituted TBsmr obtained by freeze-fracture electron microscopy, it could be shown that lipids affect the function and the oligomeric state of the protein, and that the TBsmr dimer is the minimal functional unit necessary for transport. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. In Chapter 5, the existence of such a substrate-transporter complex for the TBsmr and its substrate EtBr could be shown. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. The measurements have shown the formation of a pH-dependant, transient substrate-protein complex between binding and release of EtBr. This state was further characterized by determining the Kd, by inhibiting EtBr transport through titration with non-fluorescent substrate and by fluorescence anisotropy measurements. The findings support a model with a single occluded intermediate state in which the substrate is highly immobile. Liquid-state NMR is a useful tool to monitor protein-ligand interactions by chemical shift mapping and thus identify and characterize important residues in the protein which are involved in substrate binding. In agreement with previous studies (Krueger-Koplin et al., 2004), the detergent LPPG was found to be highly suitable for liquid-state NMR studies of the membrane protein TBsmr and 42% of the residues could be assigned, as reported in Chapter 6. However, no specific interactions with EtBr were found. This observation was confirmed by LILBID mass spectrometry which showed that TBsmr was predominantly in the non-functional monomeric state. Functional protein was prepared in proteoliposomes which can be investigated by solidstate NMR (Chapter 7). Besides the essential E13, the aromatic residues W63, Y40, and Y60 have been shown to be directly involved in drug binding and transport. Different isotope labeling strategies were evaluated to improve the quality of the NMR spectra to identify and characterize these key residues. In a single tryptophan mutant of reconstituted TBsmr W30A, the binding of ethidium bromide could be detected by 13C solid-state NMR. The measurements have revealed two populations of the conserved W63 residue with distinct backbone structures in the presence of substrate. There is a controversy about the parallel or anti-parallel arrangement of the protomers in the EmrE dimer (Schuldiner, 2007) but this structural asymmetry is consistent with both a parallel and anti-parallel topology.
- Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance (2008)
- Proteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.
- Produktion, Reinigung und Charakterisierung von monovalenten Kation/Proton - Antiportern (2007)
- Eine wichtige Klasse von Membranproteinen ist die der aktiven sekundären Transporter. Diese Proteine werden in allen Spezies gefunden und verwenden einen Gradienten von löslichen Substanzen, um den Transport von Substraten voran zu treiben. Dieser Transportprozess ist essentiell, um die chemische Zusammensetzung des Zytoplasmas, wie Kalium- oder Natriumkonzentration von der des umgebenden Milieus unterschiedlich zu halten. Die Konzentration von K+ und Na+ in der Zelle sind wichtig für ein konstantes Zellvolumen, für die pH-Homöostase, für die Erregbarkeit von Nervenzellen und füür die Akkumulierung von Zuckern und Aminosöuren über Kotransportsysteme. In Bakterien wie Escherichia coli wird mit der Oxidation von Substraten durch die Elektronentransportkette ein Protonengradient und gleichzeitig eine Potentialdifferenz erzeugt. Ein Beispiel für einen sekundären Transporter, der diese Potentialdifferenz ausnutzt ist der Na+/H+-Antiporter NhaA, einer der am besten untersuchten Antiporter aus E. coli (Hunte, Screpanti et al. 2005). Dieser Antiporter ist essentiell für die Fähigkeit von Bakterien im alkalischen pH-Bereich zu überleben. Auch bei Säugetieren, sind die Isoformen der humanen Natrium/Protonen-Antiporter SLC9A1-SLC9A8 (NHE1-8) unentbehrlich für eine Reihe physiologischer Prozesse. So wird über die Antiporter-Aktivität nicht nur der Säure-Base-Haushalt und das Verhältnis des Zellvolumens zur Menge an Elektrolyten reguliert, Antiporter spielen ebenso eine wichtige Rolle bei der Adhäsion, Migration und Proliferation der Zelle (Orlowski and Grinstein 2004). Anomalien in diesem Bereich sind charakteristisch für maligne Zellen. Die Rolle von NHE1 in der Entwicklung von Tumoren ist daher ein wichtiger Ansatzpunkt für die Entwicklung von Krebsmedikamenten. Im Herz ist NHE1 die dominierende Isoform und wird damit zu einem pharmakologisch wertvollen Zielprotein (Malo and Fliegel 2006). Struktur und Mechanismus der meisten Antiporter ist bis dato jedoch noch nicht bekannt. Neben den klassischen Methoden der Pharmaentwicklung wird die strukturbasierende Wirkstoffentwicklung immer wichtiger um effiziente Medikamente ohne Nebenwirkung zu herzustellen. Hierfür werden jedoch 3D-Strukturen von Proteinen, sowie genaue Kenntnisse von deren Mechanismus benötigt. Zieht man in Betracht, dass 70% aller bis jetzt entwickelten Medikamente als Ziel ein Membranprotein haben, wird die Notwendigkeit klar, eine möglichst große Anzahl von Membranproteinstrukturen verfgbar zu haben. Wie bereits erwähnt ist die Klasse der monovalenten Kation/Proton-Antiporter aufgrund ihrer vielfältigen Aufgaben, eine äußerst wichtige Zielgruppe für die strukturbasierende Wirkstoffentwicklung. Die große Anzahl an entschlüsselten Genomen eröffnet hier ein breites Forschungsfeld füür die Strukturbiologie. In dieser Arbeit wurden daher Techniken und Methoden aus Hochdurchsatz-orientierten Strukturgenomikprojekten übernommen, um eine große Anzahl von Zielproteinen in ausreichender Menge für die funktionelle Charakterisierung und für die Kristallisation zu produzieren. Als Zielorganismen wurden Salmonella typhimurium LT2, Helicobacter pylori 26695, Aquifex aeolicus VF5 und Pyrococcus furiosus ausgewählt. Die Grundlage dieser Entscheidung hierfür waren die humanpathogenen Eigenschaften der beiden zuerst genannten Organismen und die Hyperthermophilie der beiden letzteren. Dadurch konnten sowohl klinische Anwendungsmöglichkeiten, als auch die potentiell höhere Stabilität der hyperthermophilen Proteine genutzt werden. Als Proteinzielgruppe wurden die monovalenten Kation/Proton-Antiporter aus allen 4 Organismen ausgewählt. Des Weiteren wurden Antiporter zweier eukaryotischer Systeme, Saccharomyces cerevisiae und Homo sapiens in die Zielproteingruppe aufgenommen. In dieser Arbeit wurden 24 verschiedene monovalente Kation/Proton-Antiporter untersucht. Von diesen 24 Zielproteinen konnten 12 in Expressionsvektoren kloniert und produziert werden. Von diesen 12 Antiportern konnten die Zielproteine STM0039 (STNhaA), HP1552 (HPNhaA), STM1556 (NhaC) und PF2032 (NhaC) in einer für die Kristallisation ausreichenden Homogenität und Ausbeute gereinigt werden. Mit der Ausnahme von HP1552 ist bis heute in keiner Veröffentlichung über diese Zielproteine berichtet worden. Durch Komplementationsexperimente mit dem E. coli-Deletionsstamm EP432 konnten eine Reihe von Zielproteine (STM0039, HP1552, PF2032, Aq_2030, STM1806, STM1556) bezüglich ihrer Fähigkeiten zum Na+/H+-Antiport untersucht werden. Die Ziel-proteine STM0039, STM1556 und HP1552 konnten zum ersten Mal kloniert, produziert, gereinigt und anschlieáen in Liposomen rekonstitutiert werden.Weiterhin konnte durch SSM-Messung die pH-Regulation der Zielproteine STM0039 und HP1552 gezeigt werden. Im Gegensatz zu bisherigen Literaturangaben ist HP1552 im pH-Bereich von pH 6 bis 8,5 nicht konstitutiv aktiv, sondern erfährt eine ähnliche Aktivierung wie STM0039 oder ECNhaA. STM0039 lässt sich zudem durch 2-Aminoperimidin inhibieren. Für STM0039 konnten die ersten Proteinkristalle der inaktiven Konformation bei pH 4 erzeugt werden. Weiterhin wurde in dieser Arbeit ein gegen das Zielprotein STM0039 gerichtetes scFV-Antikörperfragment (F6scFv) eingehend charakterisiert. Durch die Ko-Kristallisation des Antikörperfragments F6scFv mit STM0039 konnten die ersten 3 dimensionalen Kristalle in einer aktiven Proteinkonformation bei pH 7,5 erzeugt werden. Neben den bereits verfeinerten Kristallisationsbedingungen für das Zielprotein STM0039 wurden erfolgreich erste Kristallisationsbedingungen für STM0086 und PF2032 gefunden. Es wurde eine Vielzahl von Produktions- und Reinigungsprotokollen füür die Zielproteine etabliert. Dadurch ist der Grundstein füür weitergehende Charakterisierungs- und Kristalli-sationsexperimente gelegt. Die in dieser Arbeit etablierte Kombination von Hochdurch-satzmethoden mit klassischen Vorgehensweisen zur Proteincharakterisierung lassen sich leicht auf anderen Membranproteinklassen bertragen und die Geschwindigkeit der ver-schiedenen Schritte bis zur Strukturlösung stark beschleunigen.
- Purification and characterisation of the respiratory supercomplex III/IV from Corynebacterium glutamicum and phospholipid analysis of membrane proteins (2008)
- The respiratory chain is composed of protein complexes residing in the inner mitochondrial membrane of eukaryotes or in the cytoplasmic membrane of prokaryotes. This cellular energy converter transforms a redox potential stored in low potential substrates into an electrochemical potential across the respective membrane. Typical respiratory chains contain the complexes I, II, III and IV named according to their sequence in the respiratory chain reaction. Electrons of low potential substrates enter at complex I or II and are passed via complex III to complex IV where they are transferred to oxygen. The transport of electrons between the complexes is mediated by small electron shuttles like quinol or cytochrome c. Two different models describe their exchange either by (1) random collision of freely diffusible electron shuttles and membrane protein complexes or (2) arrangement of the complexes in supercomplexes enabling direct channeling of electron shuttles. In the Gram positive bacterium Corynebacterium glutamicum, the complex III to complex IV electron shuttle cytochrome c is not diffusible but a covalently bound part of the diheme cytochrome subunit QcrC of complex III. Therefore, the complexes III and IV have to form a supercomplex for electron transduction. The aim of this thesis was to purify and characterise this obligatory supercomplex III/IV of C. glutamicum. To gain sufficient biomass of C. glutamicum as starting material for purification, a phosphate buffered minimal medium was developed that enabled yield of total 120 g wet cell mass (38 g dry mass) in 12 L (6×2 L) shaking cultures. The determined conversion factor of glucose into biomass was 0.46 g/g indicating an intact respiratory chain. The yield was increased by bioreactor cultivation to ~690 g wet cell mass (~220 g dry mass) in ~10 L culture volume. A previously described homologous expression system was applied that produces the complex IV subunit CtaD with a fused Strep-tag II to facilitate purification. Affinity purifications using the Strep-tag II affinity to Strep-Tactin resin yielded a mixture of complexes and supercomplexes. Two supercomplex III/IV versions named supercomplex A and B and free complex IV were identified in this mixture by size exclusion chromatography, redox difference spectroscopy and two dimensional polyacrylamide gel electrophoresis including blue native polyacrylamide electrophoresis. The here presented downscaled blue native polyacrylamide electrophoresis method with analysis times of ~1 h enabled efficient screening of factors influencing the stability of supercomplex III/IV. The screening resulted that the integrity of supercomplex III/IV is preserved by using neutral detergents at minimal detergent to protein ratios for solubilisation and low detergent concentrations for purification and storage slightly above the required critical micellar concentration. Furthermore, pH <=7.5 is required for stability of supercomplex III/IV. Large biomass yields enabled upscaling of supercomplex III/IV affinity purification. Application of the identified stability conditions resulted in affinity purified samples free of supercomplex B. The major component supercomplex A was efficiently separated from residual free complex IV by preparative size exclusion chromatography. Concentration of purified supercomplex A by ultracentrifugation resulted in integrity of the supercomplex for several days at 4 °C. Purified supercomplex A contains ten different previously described subunits. The heme content of supercomplex A relative to the protein mass is heme A: 6.0 μmol/g, heme B: 6.5 μmol/g, and heme C: 5.8 μmol/g determined by redox difference spectroscopy and biochemical protein quantification. This indicates an equimolar ratio of complex III and complex IV in supercomplex A. Supercomplex A has quinol oxidase activity that is inhibited by stigmatellin or sodium azide. The turnover number of transferred electrons per complex III monomer is 148 s−1 at 25° C. The homogeneity and stability of the prepared supercomplex A enabled the growth of threedimensional crystals of up to 0.1 mm in length. Their composition of supercomplex A was verified by redox difference spectroscopy of intact crystals and blue native polyacrylamide electrophoresis of dissolved crystals. The crystals diffracted X-rays corresponding to a resolution of ~10 Å. Electron microscopy of negative stained samples revealed the uniform shape of purified supercomplex A particles with dimensions of 22 × 9 nm in the view plane. Combined heme quantification, size determination, determined activity, symmetry considerations, and particle shape indicate that supercomplex A has a central dimer of complex III and two monomers of complex IV on opposite sides. This conformation is functionally reasonable because it provides each complex III monomer with one complex IV monomer as electron acceptor. Therefore, the stoichiometry of supercomplex A is most likely III2IV2. The sensitivity of supercomplex A to detergents indicated a role of phospholipids in its stability. Therefore, a method for phospholipid identification and quantification was developed that is suitable for detergent solubilised crude and purified membrane protein samples. The analysis combines separation of phospholipid classes according to their head group by normal phase high performance liquid chromatography with evaporative light scattering detection. Calibration with external standard allows quantification of phospholipid amount in the range of 0.25-12 μg. The method is verified by analysing the phospholipid content of the well characterised complex III of Saccharomyces cerevisiae. The reduction of its phospholipid content during its purification steps is monitored. The complex III sample purified to crystallisation quality contains the phospholipid content that was also observed in previously reported structures determined by X-ray crystallography. Purified stable supercomplex A from C. glutamicum revealed a large content of bound phospholipids. The main differences between intact supercomplex A and a mixture of potentially disintegrated smaller complexes is that intact supercomplex A has a doubled phosphatidic acid content and an increased phosphatidyl glycerol content. The importance of the small anionic phosphatidic acid for mediation of contacts between complexes in a supercomplex is discussed. The total phospholipid content of stable supercomplex A is sufficient for a complete belt surrounding the supercomplex in the membrane plane. This indicates that also all essential internal phospholipid binding positions are occupied and potentially stabilise supercomplex A.
- X-ray structure of the Na+-coupled Glycine-Betaine symporter BetP from Corynebacterium glutamicum (2009)
- Cellular membranes are important sites of interaction between cells and their environment. Among the multitude of macromolecular complexes embedded in these membranes, transporters play a particularly important role. These integral membrane proteins perform a number of vital functions that enable cell adaptation to changing environmental conditions. Osmotic stress is a major external stimulus for cells. Bacteria are frequently exposed to either hyperosmotic or hypoosmotic stress. Typical conditions for soil bacteria, such as Corynebacterium glutamicum, vary between dryness and sudden rainfall. Physical stimuli caused by osmotic stress have to be sensed and used to activate appropriate response mechanisms. Hypoosmotic stress causes immediate and uncontrolled influx of water. Cells counteract by instantly opening mechanosensitive channels, which act as emergency valves leading to fast efflux of small solutes out of the cell, therebydiminishing the osmotic gradient across the cell membrane. Hyperosmotic stress, on the other hand, results in water efflux. This is counterbalanced by an accumulation of small, osmotically active solutes in the cytoplasm, the so-called compatible solutes. They comprise a large variety of substances, including amino acids (proline), amino acid derivatives (betaine, ectoine), oligosaccharides (trehalose), and heterosides (glucosylglycerol). Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by facilitating the inward translocation of compatible solutes across the cell membrane, to restore normal hydration levels. This work presents the first X-ray structure of a member of the Betaine-Choline-Carnitine-Transporter (BCCT) family, BetP. This Na+-coupled symporter from Corynebacterium glutamicum is a highly effective osmoregulated and specific uptake system for glycine-betaine. X-ray structure determination was achieved using single wavelength anomalous dispersion (SAD) of selenium atoms. Selenium was incorporated into the protein during its expression in methione auxotrophic E. coli cells, grown in media supplemented with selenomethionine. SAD data with anomalous signal up to 5 Å led to the detection of 39 selenium sites, which were used to calculate the initial electron density map of the protein. Medium resolution and high data anisotropy made the structure determination of BetP a challenging task. A specific strategy for data anisotropy correction and a combination of various crystallographic programs were necessary to obtain an interpretable electron density map suitable for model building. The crystal structure of BetP shows a trimer with glycine-betaine bound in a three-fold cation-pi interaction built by conserved tryptophan residues. The bound substrate is occluded from both sides of the membrane and aromatic side chains line its transport pathway. Very interestingly, the structure reveals that the alpha-helical C-terminal domain, for which a chemo- and osmosensory function was elucidated by biochemical methods, interacts with cytoplasmic loops of an adjacent monomer. These unexpected monomer-monomer interactions are thought to be crucial for the activation mechanism of BetP, and a new atomic model combing biochemical results with the crystal structure is proposed. BetP is shown to have the same overall fold as three unrelated Na+-coupled symporters. While these were crystallised in either the outward- or inward-facing conformation, BetP reveals a unique intermediate state, opening new perspectives on the alternating access mechanism of transport.
- Improving methods for the study of membrane proteins by solid-state NMR (2009)
- Solid state NMR is a emerging method for the study of membrane proteins, which has received much interest in recent years. Limiting the study of many pharmacologically relevant targets, are the often long measuring times, required to obtain especially higher dimensional solid state NMR spectra of good quality. To address this problem, multiple methods where developed in this work, which can be categorized into two groups. The first set of methods aims at the quality of certain spectra, by implementing a spectral filter, which increases the fidelity of the measured data. The second set of methods, addresses the problem of long measuring times directly, by increasing the sensitivity per unit time, as could be shown, for example, on homo- and heteronuclear singlequantum-singlequantum correlation experiments. The gains in measuring time for the latter group of methods are typically in the order of 2-3, but some experiments allow multiple methods to be employed simultaneously, which can lead to a decrease in measuring time of a factor of up to 8. It is important to mention, that none of the methods introduced in this work require any equipment in addition to the conventional setup present in most sold state NMR laboratories and no changes or addition to the samples under study are required. Therefore the gains reported in this work come at no extra cost and require only minimal implementation effort on the side of the user.
- Pulsed EPR characterization of membrane transport protein complexes (2012)
- Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems and recently is increasingly applied to membrane proteins. However, after reconstitution of labeled proteins into liposomes, spin labels often exhibit a much faster transversal relaxation (Tm) than in detergent micelles, thus limiting application of the method in lipid bilayers. In the first part of the thesis, optimization of transversal relaxation in phospholipid membranes was systematically investigated by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin aggregation and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples with low local concentrations does deuteration of acyl chains and buffer significantly prolong Tm. In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect Tm, either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions. In the second part of the thesis, inhomogeneous distribution of spin-labels in detergent micelles has been studied. A common approach in PELDOR is measuring the distance between two covalently attached spin labels in a macromolecule or singly-labeled components of an oligomer. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, it is shown that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces. In the third part, PELDOR has been utilized to study the structural flexibility of the Toc34 GTPase homodimer, a preprotein receptor of the translocon of the outer envelope of chloroplasts (TOC). Toc34 belongs to GAD subfamily of G-proteins that are regulated and activated by nucleotide-dependent dimerization. However, the function of Toc34 dimerization is not yet fully understood. Previous structural investigations of the Toc34 dimer yielded only marginal structural changes in response to different nucleotide loads. PELDOR revealed a nucleotide-dependent transition of the dimer flexibility from a tight GDP to a flexible GTP-loaded state. Substrate-binding stabilizes the dimer in the transition state mimicked by GDP-AlFx, but induces an opening in the GDP or GTP-loaded state. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which represents the regulatory mode for dimerizing GTPases. In the fourth part of the thesis, conformational flexibility and relative orientation of the N-terminal POTRA domains of a cyanobacterial Omp85 from Anabaena sp. PCC 7120, a key component of the outer membrane protein assembly machinery, were investigated by PELDOR spectroscopy. Membrane proteins of the Omp85-TpsB superfamily are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. It has been suggested that the N-terminal POTRA domains (P1 and P2) might have functions in substrate recognition. Molecular dynamics (MD) simulations predicted a fixed orientation for P2 and P3 and a flexible hinge between P1 and P2. The PELDOR distances measured between the P2 and P3 POTRA domains are in good agreement with the structure determined by X-ray, and compatible with the MD simulations suggesting a fixed orientation between these domains. PELDOR constraints between the P1 and P2 POTRA domains imply a rather rigid structure with a slightly different relative orientation of these domains compared with the X-ray structure. Moreover, the large mobility predicted from MD is not observed in the frozen solution. The PELDOR results further highlight the restricted relative orientation of the POTRA domains of the Omp85-TpsB proteins as a conserved characteristic feature that might be important for the processive sliding of the unfolded substrate towards the membrane.