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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.
Channelrhodopsin-2 (ChR2) is a light-gated cation selective channel from the unicellular alga Chlamydomonas reinhardtii, which is involved in phototaxis and photophobic responses. As other rhodopsins, ChR2 comprises a seven-transmembrane helix (TMH) motif and a retinal as the light-sensitive chromophore. The chromophore is covalently attached via a protonated Schiff base to the conserved lysine residue Lys257 located in TMH7. Based on its primary sequence and the all-trans configuration of the retinal in the ground state, ChR2 is assigned to the type I rhodopsins, also referred to as microbial-type rhodopsins. Upon light activation, the retinal isomerizes from the all-trans to the 13-cis form. This photoisomerization, which is accompanied by conformational changes of the protein, eventually leads to the opening of the channel and cation translocation. Cation flux during the conductive state leads to depolarization of the cell membrane and subsequent triggering of action potentials when expressed in neurons. Therefore, ChR2 has become the most versatile optogenetic tool, enabling a non-invasive investigation of neural circuits at high spatial and temporal resolution. With the rapidly increasing importance of ChR2 as a tool in neurobiology and cell biology, structural information is the prerequisite to an unambiguous understanding of the molecular mechanisms of this unique light-activated ion channel. The coupling between isomerization and structural alterations is well understood for other microbial-type rhodopsins, like bacteriorhodopsin (bR), halorhodopsin (HR) and sensory rhodopsin II (SRII). In case of ChR2, the first data on light-induced conformational changes came from spectroscopic studies and structural information is still missing. However, in order to fully understand the mechanism of light transduction by ChR2, it is necessary to determine the changes in the protein structure at specific steps in the photocycle.
By the time I started my PhD thesis, there was no structural information of ChR2 available. Therefore, the objective of this thesis was to obtain structural information of the transmembrane domain containing the first 315 amino acids of ChR2 by cryo electron crystallography. Besides revealing the structure of membrane proteins, cryo-EM of two-dimensional (2D) crystals is ideal for investigating conformational changes in membrane proteins induced by different stimuli. Therefore, the second objective of my thesis was the investigation of light-induced conformational changes in the slow C128T ChR2 mutant. The ~1,000 times longer lifetime of the open state of the C128T mutant compared to the wild-type allowed to trap different intermediates that accumulate during the photocycle.
In 2012, the X-ray structure of a channelrhodopsin-1/channelrhodopsin-2 chimaera (C1C2) at 2.3 Å resolution in the closed dark-adapted state was published (Kato et al., 2012). The structure revealed the essential molecular architecture of C1C2, including the retinal-binding pocket and the putative cation conduction pathway. Together with biochemical, spectroscopic, mutagenesis experiments, and the high-resolution model, some functionally important residues of ChR2 have been identified. However, unambiguous explanation of the molecular determinants that contribute to activation (gating) and transport were still mostly unknown.
RESULTS AND CONCLUSIONS
The first half of my theses dealt with 2D crystallization of ChR2. I succeeded in obtaining 2D crystals of ChR2 of four different types, which differed in size, crystal packing, crystal contacts and resolution, yielding structure factors up to 6 Å resolution. The crystals were grown by reconstituting the protein with different lipids at various lipid-to-protein ratios. The best crystals formed with the synthetic lipid DMPC and EPL upon detergent removal by dialysis. The projection maps calculated from these crystals revealed the overall structure of C128T ChR2 at 6 Å resolution and were published in 2011 (Müller et al., 2011). Surprisingly, ChR2 was found to be a dimer in all crystal types. The ChR2 dimer was stable both in detergent solution and in the presence of lipids for 2D crystallization. The monomers clearly showed the expected densities for the seven TMHs.
The arrangement of the ChR2 dimers on the four 2D lattices was different. However, comparison of the individual rojection maps revealed no significant differences within the ChR2 interface in the four crystal forms. The observation that the structure of the dimer was the same in all four crystal forms and in different lipids suggested strong specific contacts between the two protomers and implied that the protein was also dimeric in the native membrane. These findings were in agreement with Western blot analysis of plasma membranes from oocytes expressing ChR2 and laser-induced liquid bead ion desorption mass spectrometry, which both showed ChR2 as a dimer. The unusual stability of the ChR2 dimer contrasts with other microbial rhodopsins, which exist in different oligomeric states, i.e. monomers, trimers or dimers. These observations raised the question whether the functional unit is the monomer or the dimer.
The comparison of the projection map of the light-driven proton pump bR at the same resolution showed similar overall dimensions. Based on this comparison, the densities which became evident in the ChR2 projection maps could be assigned to the corresponding seven densities in bR. The shape of the densities near the dimer interface suggested that TMHs 2, 3, and 4 are oriented more or less perpendicular to the membrane plane, while the other four helices appear to be more tilted, as in bR.
Based on the high-resolution bR structure and the projection structures obtained, I have built a homology model. On the basis of this homology model, several residues found in the dimer interface were selected for mutational studies in order to disrupt the dimer interface.
The investigation of light-induced conformational changes in C128T ChR2 was the second part of my thesis. I designed an experimental setup for trapping light-induced conformational changes in C128T ChR2. In addition, I optimized the sample preparation in a way that the different illumination conditions did not alter the quality of the crystals. I have trapped two different functional states, namely the conductive open state and the non-conductive closed dark-adapted state.
In order to visualize the location and the extent of conformational changes, projection difference maps were calculated between the open and the closed state. Visual inspection of the difference maps between the open and the two closed states revealed three difference peaks that map to the TMHs 2, 6, and 7, indicating significant and specific rearrangements of these helices. The strong pair of positive/negative peaks at TMH6 suggests an outward tilt movement of approximately 2 Å. Close comparison of similar work on bR revealed that this movement is likely to occur at the cytoplasmic end of TMH6. A second highly significant negative peak is observed at TMH7, indicating a less pronounced tilt compared to TMH6. The third negative peak at TMH2 indicates a loss of density in this region. No significant differences were recorded at the TMH1, 5 and at the dimer interface formed by TMH3 and 4.
I succeeded in trapping and characterizing the open and closed state in the photocycle of ChR2 and could demonstrate that the transition from the closed to the open state is linked to significant light-induced tilt movements of TMH6 and 7, plus a loss of order in TMH2. These conformational changes are likely to create a large water-filled conducting pore, which seems to be required for the conductance of up to 2,000 ions per photocycle. The previously mentioned spectroscopic studies support the difference structures I obtained. This approach sets the stage for studying structural changes accompanying the formation and decay of other photocycle intermediates in ChR2. Future studies will aim at three-dimensional maps of the open and closed state at higher resolution.
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.