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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.
The increasing resistance of almost all pathogenic bacteria to antibiotics (multidrug resistance) causes a severe threat to public health. The mechanisms underlying multidrug resistance include the induced over expression of multidrug transporters which extrude a variety of lipophilic and toxic substrates in an energy dependent fashion through the membrane out of the cell. These proteins are found in all transporter families. The work described in this thesis is dedicated to drug-proton antiporters from the small multidrug resistance (SMR) family. These efflux pumps with just four transmembrane helices per monomer are so far the smallest transporters discovered. Their oligomeric state, topology, three dimensional structure, catalytic cycle and transport mechanism are still rather controversial. Therefore, the aim of this thesis was to directly address these questions for the small multidrug resistance proteins Halobacterium salinarium Hsmr and Escherichia coli (E. coli) EmrE using a number of biophysical methods such as NMR, transport assays, mass spectrometry and analytical ultracentrifugation. Especially the work on Hsmr has been challenging due to the halophilic nature of this protein. In Chapter 1, key questions and the most important biophysical techniques are introduced followed by Material and Methods in Chapter 2. Depending on experimental requirements, cell free or ‘classical’ in vivo expression has been used for this thesis. Cell free expression as an option for the production of small multidrug transporters has been explored in Chapter 3. It has been possible to produce the SMR family members Hsmr, EmrE, TBsmr and YdgF in vitro. The expression of Hsmr was investigated in more detail under different experimental conditions. Hsmr was either refolded from precipitate or maintained in a soluble form during expression in the presence of detergents and liposomes. Furthermore, amino acids for which no auxotrophic strains were available could be labelled successfully. This expression system has been also used for preparing labelled samples of EmrE as described in Chapter 9. In vivo in E. coli expression of Hsmr, as described in Chapter 4, provided large amounts of proteins if fermenter production was used. Uniform labelling and selective unlabelling with stable isotopes (13C, 15N) for NMR spectroscopy was achieved in vivo in a more efficient and cost effective manner than using the cell free approach for this protein. Hsmr could be purified successfully from both in vitro and in vivo expression media. Hsmr is expressed in vivo and in vitro with N-terminal formylation. The Nterminal formylation is unstable and Hsmr in the presence of low salt concentrations was amenable to N-terminal degradation. It was found that Hsmr shows longest stability in Fos-ß-choline® 12 and sodium dodecyl sulphate, but best reconstitution conditions were found, when dodecyl maltoside is used and exchanged with Escherichia coli lipids. A molar protein lipid ratio of 1 to 100, amenable to solid state nuclear magnetic resonance, has been achieved. Sample homogeneity was shown by freeze fracture electron microscopy. The oligomeric state of Hsmr in detergent has been assessed by SDS PAGE, blue native PAGE, size exclusion chromatography, analytical ultracentrifugation and laser induced liquid bead ion desorption mass spectrometry (LILBID) as described in Chapter 5. A concentration and detergent dependent monomer-oligomer equilibrium has been found by all methods. The activity of Hsmr under the sample preparation conditions used here was shown using radioactive and fluorescence binding as well as fluorescence and electrochemical transport assays (Chapter 6). For transport studies, a stable pH gradient was generated by co-reconstitution of Hsmr with bacteriorhodopsin and subsequent sample illumination. Based on the observed long term stability of Hsmr in Fos-ß-choline® 12 and sodium dodecyl sulphate, liquid state NMR experiments were attempted in order to assess the correct folding of Hsmr in detergent micelles (Chapter 7). 1D proton and 2D HSQC spectra of U-15N Hsmr revealed a poor spectral dispersion, low resolution and only a small number of peaks. These are at least partly due to long rotational correlation times of the large protein detergent complex. This problem has been overcome by applying solid-state NMR to Hsmr reconstituted into E. coli lipids (Chapter 8). Uniform 13C labelled samples were prepared and two dimensional proton-driven spin diffusion and double quantum-single quantum correlation spectra were acquired successfully. Unfortunately, the spectral resolution was not yet sufficient for further structural studies. Reasons for the observed linebroadening could be structural heterogeneity or molecular motions which interfere with the NMR timescale. Therefore, the protein mobility has been probed using static 2H solid state NMR on Ala-d3-Hsmr. It could be shown, that parts of Hsmr are remarkably mobile in the membrane and that this mobility can be limited by the addition of the substrate ethidium bromide. Ethidium bromide as well as tetraphenylphosphonium (TPP+) is typical multidrug transporter substrates. The membrane interaction of TPP+ in DMPC membranes has been resolved by 1H MAS NMR. It was found that it penetrates into the interface region of the lipid bilayers and therefore behaves like many other transporter substrates adding to the hypothesis that the membrane could act as a pre-sorting filter. Finally, Chapter 9 is dedicated to the characterisation of the essential and highly conserved residue Glu-14 in EmrE by solid-state NMR. In order to avoid spectral overlap, the single Glu EmrE E25A mutant was chosen instead of the wildtype. The protein has been produced in vitro to take advantage of reduced isotope scrambling in the cell free expression system as verified by analytical NMR spectroscopy. Correct labelling of EmrE was tested by MALDI-TOF and solid-state NMR. The dimeric state of DDM solubilised EmrE has been probed by LILBID. The labelled protein was reconstituted into E. coli lipids to ensure a native membrane environment. Activity was determined by measuring ethidium bromide transport. Freeze fracture EM revealed very homogeneous protein incorporation even after many days of MAS NMR experiments. 2D 13C double quantum filtered experiments were used to obtain chemical shift and lineshape information of Glu-14 in EmrE. Two distinct populations were found with backbone chemical shift differences of 4 - 6 ppm which change upon substrate binding. These findings indicate a structural asymmetry at the assumed dimerisation interface and are discussed in the context of a model for shared substrate/proton binding. These studies represent the first successful use of cell free expression to prepare labelled membrane proteins for solid-state NMR and allow for the first time an NMR insight into the binding pocket of a multidrug efflux pump.
In this study, a portable electronic nose (E-nose) prototype is developed using metal oxide semiconductor (MOS) sensors to detect odors of different wines. Odor detection facilitates the distinction of wines with different properties, including areas of production, vintage years, fermentation processes, and varietals. Four popular machine learning algorithms—extreme gradient boosting (XGBoost), random forest (RF), support vector machine (SVM), and backpropagation neural network (BPNN)—were used to build identification models for different classification tasks. Experimental results show that BPNN achieved the best performance, with accuracies of 94% and 92.5% in identifying production areas and varietals, respectively; and SVM achieved the best performance in identifying vintages and fermentation processes, with accuracies of 67.3% and 60.5%, respectively. Results demonstrate the effectiveness of the developed E-nose, which could be used to distinguish different wines based on their properties following selection of an optimal algorithm.
Succinate:quinone oxidoreductases (SQORs) are integral membrane protein complexes, which couple the two-electron oxidation of succinate to fumarate (succinate → fumarate + 2H+ + 2e-) to the two-electron reduction of quinone to quinol (quinone + 2H+ + 2e- → quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol. In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as complex II of the aerobic respiratory chain or as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle, catalyzes the oxidation of succinate by ubiquinone, which is mildly exergonic under standart conditions and not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather menaquinone, a quinone with significantly lower oxidation-reduction (“redox”) midpoint potential. In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions. Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in vivo. Based on experimental evidence obtained on whole cells and purified membranes, it had previously been proposed that the SQR from Gram-positive bacteria supports this reaction at the expense of the protonmotive force, Δp. Nonetheless, it has been argued that the observed Δp dependence is not associated specifically with the activity of SQR because the occurrence of artifacts in experiments with bacterial membranes and whole cells can not be fully excluded. Clearly, definitive insight into the mechanism of catalysis of this intriguing reaction required a corresponding functional characterization of an isolated, membranebound SQR from a Gram-positive bacterium. The first aim of the present work addresses the question if the general feasibility of the energetically uphill electron transfer from succinate to menaquinone is associated specifically to a single enzyme complex, the SQR. The prerequisite to achieve this goal was stable preparation of this enzyme.
The universal biological energy currency adenosine triphosphate (ATP) is synthesized by the F1Fo-ATP synthase in most living organisms. The overall structure and function of F-type ATPases is conserved in the different organisms. The F1Fo-ATP synthase consist of two domains; the soluble F1 complex has the subunit stoichiometry α3β3γδε and the membrane embedded Fo complex consists of subunits ab2c10-15 in its simplest form found in bacteria. F1 and Fo both function as reversible rotary motors that are connected by a central stalk (γε) and a peripheral stalk (b2δ).
For ATP synthesis, the electrochemical energy formed by a proton or sodium ion gradient is required. The ion translocation across the Fo subcomplex induces torque in the motor part of the enzyme (cnγε), which causes conformational changes in the α3β3 domain leading to ATP synthesis from ADP and inorganic phosphate (Pi) catalyzed in the β-subunits. ATP hydrolysis causes a reverse torque in the Fo subcomplex triggering uphill ion translocation from cytoplasm to periplasm, and the enzyme functions as an ion pump.
The ATP synthesis mechanism is well understood, since several high-resolution structures of F1 are available. In contrast, the ion translocation mechanism across the membrane, mediated by the Fo subcomplex, is not understood in its structural detail.
Subunit a and the c-ring form an ion pathway, but subunit b is needed to form an active ion translocation pathway in both H+- and Na+-dependent systems. Several high-resolution structures of c-rings have provided insights in the ion translocation mechanism. The different ion translocation models based on biochemical, biophysical and structural analysis are in agreement in the fact that ions are translocated through a periplasmic ion access pathway in subunit a to the middle of the membrane and there to the binding site of a c-subunit. After almost a whole rotation of the c-ring the ion returns into the a-c interface, where it can be released to the cytoplasm. In the different models the cytoplasmic access pathway has been proposed to be located in subunit a, at the a-c interface or within the c-ring. The driving force of torque generation has been proposed to be the pH gradient or membrane potential. Several biochemical studies show that a conserved arginine in helix four of subunit a (R226 in Ilyobacter tartaricus or R210 in Escherichia coli)plays a critical role in the ion translocation. The arginine has been proposed to function as an electrostatic separator between the cytoplasmic and periplasmic pathways and as a mediator of the ion exchange into the c-ring ion-binding site.
Structural data of a related enzyme (V1Vo-ATPase from Thermus thermophilus) has provided insight into the helical arrangement of the ion translocating subunits I and Lring (related to subunit a and the c-ring). These structures indicated a small interface between subunit I and the L-ring, and two four-helix bundles in the N-terminal domain of subunit I were proposed to build the periplasmic and cytoplasmic ion pathways. To comprehend the ion-translocation and torque generation mechanism in F1Fo-ATP synthase, structural data of an intact a-c complex is needed.
The goal of this work was to obtain structural data of subunit a, most preferably in a complex with the c-ring or additionally with subunit b. Therefore, a new purification procedure for the I. tartaricus Fo-subcomplex, heterologously expressed in E. coli cells, was established. The purified Fo was characterized biochemically and by Laserinduced liquid bead ion desorption mass spectrometry (LILBID-MS). These analyses showed that pure and completely assembled Fo containing all its subunits in the correct stoichiometry (ab2c11) was obtained. The purified Fo complex was stable at 4°C for several months and at room temperature in the presence of lipids for several weeks. A lipid analysis was performed by thin-layer chromatography (TLC) to investigate the qualitative lipid composition of I. tartaricus whole lipid extract and various I. tartaricus F1Fo isolates. The whole lipid extract contained PC, PG and PE lipids and probably cardiolipin. PC, PG and PE lipids were bound to wild type I. tartaricus F1Fo, whereas recombinant I. tartaricus F1Fo did not have any bound lipids, but was able to bind the synthetic lipids POPC and POPG if they were provided during the purification.
For subsequent structural studies the purified Fo was subjected to two-dimensional (2D) crystallization trials. Vesicles and sheets tightly packed with protein and crystals with a rare plane group for I. tartaricus c11 (p121) were obtained. The c-ring was visible in the CCD images, and immunogold-labeling revealed the presence of the His-tagged a-subunit in the reconstituted vesicles. Furthermore, atomic force microscopy (AFM) imaging showed protein densities next to the c-rings, which protruded less from the membrane (0.4±0.1 nm) than the c-ring (0.7±0.1 nm). These protein densities presumably belonged to subunit a.
Cryo-electronmicroscopy (cryo-EM) was used to collect data of the p121 crystals and a merged projection density map was calculated to 7.0 Å resolution. The unit cell of the crystals (81 × 252 Å) contained two asymmetric units with three c-rings in each and next to the c11-rings new prominent densities were visible. In each extra density up to 7 transmembrane helices were visible, belonging to the stator subunit a and/or subunit b. To elucidate whether there are conserved elements in the three extra densities non-crystallographic averaging was applied using a single-particle approach.
Six possible arrangements for the c-rings and the extra densities were identified and used for the averaging. The extra densities were enhanced only in one of the possible arrangements. The average showed a four-helix bundle and a fifth helix in close proximity to the c-ring. Two more helices were present in each position but their position was ambivalent. The data obtained in this work provides the first insight in the helical arrangement in the a-c interface of F1Fo-ATP synthase.
ATP synthases are multi-subunit membrane enzymes, which utilize the energy stored in a transmembrane electrochemical ion gradient to produce adenosine-5´-triphosphate (ATP), the universal energy carrier in biological systems. Research on these important enzymes goes back more than 50 years and has produced innumerable studies. The F-type ATP synthase consists of two functionally distinct, but tightly coupled subcomplexes, the water-soluble F1 and the membrane-embedded Fo complex. In its simplest form, F1 consists of five different subunits with a stoichiometry of α 3β3γδε, and harbors three catalytic centers in the α 3β3-headpiece, while Fo consists of three different subunits in a stoichiometry of ab2cn, where n varies between 8 to 15 depending on the species. From a mechanistic standpoint, the complex can also be divided into two different units, namely a stator, α3β3δ-ab2, and a rotor, γε-cn. The enzyme utilizes the energy stored in a transmembrane electrochemical gradient of protons, or in some cases Na+, to drive ATP synthesis. In particular, the downhill translocation of these ions across the Fo complex drives rotation of the γε-cn unit, which is then transduced to the active centers, catalyzing the phosphorylation of adenosine-5`-diphosphate (ADP) with inorganic phosphate (Pi), and the release of ATP....
G-protein coupled receptors (GPCRs) are the key players in signal perception and transduction and one of the currently most important class of drug targets. An example of high pharmacological relevance is the human endothelin (ET) system comprising two rhodopsin-like GPCRs, the endothelin A (ETA) and the endothelin B (ETB) receptor. Both receptors are major modulators in cardiovascular regulation and show striking diversities in biological responses affecting vasoconstriction and blood pressure regulation as well as many other physiological processes. Numerous disorders are associated with ET dysfunction and ET antagonism is considered an efficient treatment of diseases like heart failure, hypertension, diabetes, artherosclerosis and even cancer. This study exemplifies strategies and approaches for the preparative scale synthesis of GPCRs in individual cell-free (CF) systems based on E. coli, a newly emerging and promising technique for the production of even very difficult membrane proteins. The preparation of high quality samples in sufficient amounts is still a major bottleneck for the structural determination of the ET receptors. Heterologous overexpression has been a challenge now for decades but extensive studies with conventional cell-based systems had only limited success. A central milestone of this study was the development of efficient preparative scale expression protocols of the ETA receptor in qualities sufficient for structural analysis by using individual CF systems. Newly designed optimization strategies, the implementation of a variety of CF expression modes and the development of specific quality control assays finally resulted in the production of several milligrams of ETA receptor per one millilitre of reaction mixture. The versatility of CF expression was extensively used to modulate GPCR sample quality by modification of the solubilization environment with detergents and lipids in a variety of combinations at different stages of the production process. Downstream processing procedures of CF synthesized GPCRs were systematically optimized and sample properties were analysed with respect to homogeneity, protein stability and receptor ligand binding competence. Evaluation was accomplished by an array of complementary and specifically modified techniques. Depending on its hydrophobic environment, CF production of the ETA receptor resulted in non-aggregated, monodisperse forms with sufficient long-term stability and high degrees of secondary structure thermostability. The obtained results document the CF production of the ETA receptor in two different modes as an example of a class A GPCR in ligand-binding competent and non-aggregated form in quantities sufficient for structural approaches. The presented strategy could serve as basic guideline for the production of related receptors in similar systems.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.
ncubation of class II chloroplasts of spinach with copper in the light at pH = 8 in concentrations that inhibit oxygen evolution results in the formation of a copper (II) protein complex with the photosynthetic membrane. The EPR spectra indicate that the four nearest ligands to Cu(II) consist of three oxygen atoms and one nitrogen atom. The copper (II) protein appears to be pre dominantly associated with photosystem II. The formation of this protein as measured by the EPR signal amplitude of its room temperature spectrum correlates with the inhibition of oxygen evolution and of electron transport within photosystem I. This result indicates that the inhibition of photosynthetic electron transport by copper may be due to the formation of a copper (II) chelate with a membrane protein.