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The geometric parameters of the title compound, C8H6N2O·C6H3N3O7, are in the usual ranges. The three nitro groups are almost coplanar with the aromatic picrate ring [dihedral angles 10.2 (2)°, 7.62 (16) and 8.08 (17)°]. The molecular conformation of the picric acid is stabilized by an intramolecular O-H...O hydrogen bond. The phthalazin-1(2H)-one molecules are connected via N-H...O hydrogen bonds, forming centrosymmetric dimers. Key indicators: single-crystal X-ray study; T = 173 K; mean σ(C–C) = 0.002 Å; R factor = 0.034; wR factor = 0.091; data-to-parameter ratio = 11.1.
The retinoic acid related orphan receptor alpha (RORalpha) regulates the expression of various target genes by binding to specific response elements in their promoter region. RORalpha is an interesting pharmaceutical target since it positively affects several pathophysiological processes of clinical relevance. RORalpha enhances the expression of Apo-AI protein, the major constituent of HDL, which is responsible for the cholesterol transportation. RORalpha notably contributes to the bone mineralization and generation of the extracellular bone matrix, demonstrating its involvement in osteoporosis, and by up-regulating the gene for IKBalpha, RORalpha has anti-inflammatory effects. Moreover, RORalpha is necessary for cerebellar development and the maintenance of the mammalian day-night periodicity governed by the core-clock within the suprachiasmatic nuclei. RORalpha receptors have been reported to bind cholesterol, melatonin, or to function ligand-independent. By monomeric binding to the recognition motif AGGTCA preceded by an A/T-rich sequence (ROR response element, RORE), RORalpha constitutively activates gene transcription. However, RORalpha activity is passively suppressed by its opponents RevErbalpha and RevErbbeta, which both bind to the same target sequence. ...
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.
The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
Identification of a lysosomal peptide transport system induced during dendritic cell development
(2007)
The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.
The ABC protein ABCE1, also called HP68 or RNase L inhibitor (RLI), is one of the most conserved proteins in evolution. It is universally expressed in eukaryotes and archaea, where ABCE1 is essential for life. ABCE1 plays a crucial role in translation initiation and ribosome biogenesis, however, the molecular mechanism of ABCE1 remains unclear. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines predicted to coordinate iron-sulfur (Fe-S) clusters. To analyze the function of ABCE1, the hyperthermophilic crenarchaeote Sulfolobus solfataricus was chosen as a model system. S. solfataricus ABCE1 was overexpressed homologously in S. solfataricus and heterologously in E. coli. Noteworthy, for tagged-protein production in S. solfataricus a novel expression system based on a virus shuttle vector was established. This is the first example for a successful overexpression and purification of isolated full-length ABCE1. For the first time it was shown that ABCE1 indeed bears biochemical properties of an ABC protein even though it has unique features. Remarkably, the nucleotide binding domains (NBDs) of ABCE1 bound ATP and AMP, but were functionally non-equivalent in ATP hydrolysis. Mutations of conserved residues in the second NBD led to a hyperactive ATPase, which implies an intramolecular mechanism of dimer formation. Truncation of the Fe-S cluster domains did not influence ATPase activity. The Fe-S clusters of ABCE1 were analyzed by biophysical and biochemical methods. As presented in this study, ABCE1 harbors two essential diamagnetic [4Fe-4S]2+ clusters, one ferredoxin-like cluster formed by cysteines at position 4/5/6/7 and one unique ABCE1 cluster formed by cysteines at position 1/2/3/8. ABCE1 was found to be associated with RNA after purification from S. solfataricus and bound ribosomal RNA in vitro. In addition, ABCE1 showed homo-oligomerization and appeared to form a hexameric complex of ~440 kDa, which was RNase sensitive. Archaeal ABCE1 associated with ribosomes, however, the unique Fe-S clusters of ABCE1 were not required for this interaction. Although archaeal ABCE1 assembled with ribosomes and ribosomal RNA, ABCE1 proved not to be essential for translation in S. solfataricus and did not interact with archaeal initiation factors. Nevertheless, the ABCE1 gene is one of the few genes conserved between archaea and eukaryotes and fulfills a universal task, which needs further characterization.
Analysis of coding principles in the olfactory system and their application in cheminformatics
(2007)
Unser Geruchssinn vermittelt uns die Wahrnehmung der chemischen Welt. Im Laufe der Evolution haben sich in unserem olfaktorischen System Mechanismen entwickelt, die wahrscheinlich optimal auf die Erfüllung dieser Aufgabe angepasst sind. Die Analyse dieser Verarbeitungsstrategien verspricht Einblicke in effiziente Algorithmen für die Kodierung und Verarbeitung chemischer Information, deren Entwicklung und Anwendung dem Kern der Chemieinformatik entspricht. In dieser Arbeit nähern wir uns der Entschlüsselung dieser Mechanismen durch die rechnerische Modellierung von funktionellen Einheiten des olfaktorischen Systems. Hierbei verfolgten wir einen interdisziplinären Ansatz, der die Gebiete der Chemie, der Neurobiologie und des maschinellen Lernens mit einbezieht.
Two distinct mechanisms contribute to the development of blood vessels: vasculogenesis, which is the de novo formation of vascular structures from progenitor cells, and angiogenesis, the formation of new blood vessels from pre-existing ones.
Angiogenesis is a highly ordered and carefully regulated multi-step process, during which the precise spatio-temporal interaction between endothelial and mural cells, i.e. smooth muscle cells and pericytes, is prerequisite for the formation of a functional blood vessel. The crosstalk between these two latter cell ty pes is mediated indirectly by various
secreted growth factors, and directly through cell-cell and cell-matrix interactions. The secretory epidermal growth factor-like protein 7 (EGFL7) has been implicated to
play an important role in the regulation of smooth muscle and endothelial cell recruitment and vascular tube formation. However, in-depth investigation of the underlying molecular mechanism has so far been hampered by the lack of functional recombinant EGFL7. In this study for the first time full length EGFL7 was successfully expressed as a His 6- tagged fusion protein from insect cells using the Baculovirus expression vector system. Recombinant EGFL7 was purified in a two-step protocol involving ion metal affinity chromatography and gel filtration. Furthermore, recombinant EGFL7 was
purified from human embryonic kidney EBN A 293 cells using a similar approach, allowing the production of high amounts of recombinant EGFL7 protein in its native state, with proper post-translational processing and full biological activity. Detailed analysis of the post-translational processing of recombinant EGFL7 and EGFL7-mutants revealed extensive proteolytic processing by protein convertases both at the N- and the C-terminus, the latter being prerequisite for EGFL7 secretion. Furthermore, secreted EGFL7 protein was shown to bind to the extracellular matrix and the responsible heparin-binding domain of EGFL7 was mapped to its N-terminal
portion. Purified recombinant EGFL7 protein was tested for its functionality using cell migration assays, cell proliferation studies and in vivo matrigel studies in mice. In the
modified Boyden chamber migration assay, recombinant EGFL7 proteins inhibited PDGF-BB-induced smooth muscle cell migration. Moreover, recombinant EGLF7 proteins strongly inhibited PDGF-BB-induced proliferation of smooth muscle cells, while it did not affect VEGF induced proliferation of endothelial cells. When applied in the in vivo matrigel plug assay, EGFL7 proteins induced a strong pro-angiogenic response, comparable with that of VEGF on an equimolar basis. Moreover, EGFL7 expression was strongly induced in endothelial cells in response to VEGF stimulation. These novel findings demonstrate the important function of EGFL7 in angiogenesis and are well in line with previous results. They demonstrate a cell specific action of EGFL7 on the different cell types involved in vessel formation, which is a prerequisite for a regulatory function in cell-to-cell crosstalk. Based on the results described here, the following model can be proposed: VEGF, a known strong initiator of angiogenesis, induces endothelial cell proliferation and migration, allowing the
escape from the comparatively rigid structure of a functional vessel to form an angiogenic sprout. At the same time VEGF induces the expression of EGFL7 in endothelial cells. EGFL7 is expressed, proc essed and secreted from these cells. While EGFL7 has no known effect on endothelial cells, it inhibits smooth muscle cell proliferation and migration, providing a mechanism to prevent pre-mature stabilization of the forming vessel. The availability of purified recombinant EGFL7 will be helpful in the detailed characterization of the underlying molecular mechanism of EGFL7 action, including the identification of the putative EGFL7 receptor, and will allow - together with knock-out experiments in mice - the exploration of the additional biological functions of EGFL7. Moreover, considering the strong pro-angiogenic effect of EGFL7 in vivo, it would be also of a great therapeutic interest to investigate its role in the development of tumor vasculature. The insights into these molecular mechanisms might provide a novel approach for the development of anti tumor therapies.
The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.
2,5-Diformylbenzene-1,4-diol (5) is a well-suited starting compound for the preparation of ditopic hydroquinone-based ligands. Here, we report an optimized synthesis of 5 which improves the overall yield from published 7% to 42 %. Three new ditopic Schiff base ligands, 2,5-[iPr2N(CH2)2N=CH]2 - 1,4-(OH)2-C6H2 (8), 2,5-(pyCH2N=CH)2-1,4-(OH)2-C6H2 (9), and 2,5-[py(CH2)2N=CH]2-1,4- (OH)2-C6H2 (10), have been synthesized from 5 and structurally characterized by X-ray crystal structure analysis (py = 2-pyridyl).
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
Oxidative stress attenuates the NO-cGMP pathway, e.g. in the vascular system, through scavenging of free NO radicals by superoxide O2•-, by inactivation of soluble guanylyl cyclase (sGC) via oxidation of its central Fe2+ ion, and by down-regulation of sGC protein levels. While the former pathways are well established, the molecular mechanisms underlying the latter are still obscure. Using oxidative sGC inhibitor ODQ we demonstrate rapid down-regulation of sGC protein in mammalian cells. Co-incubation with proteasomal inhibitor MG132 results in accumulation of ubiquitinated sGC whereas sGC activator BAY 58–2667 prevents ubiquitination. ODQ-induced down-regulation of sGC is mediated through selective ubiquitination of its b subunit, and BAY 58–2667 abrogates this effect. Ubiquitination of sGC-b is dramatically enhanced by E3 ligase CHIP. Our data indicate that oxidative stress promotes ubiquitination of sGC b subunit through E3 ligase CHIP, and that sGC activator 58–2667 reverts this effect, most likely through stabilization of the heme-free b subunit. Thus the deleterious effects of oxidative stress can be counter-balanced by an activator of a key enzyme of vascular homeostasis.
To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5-iodouridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE (R) chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.
An application of EPR spectroscopy that is becoming increasingly important is the measurement of distances between electron spins. Several EPR methods have been developed for this purpose, all based on measuring the dipolar coupling between two spins. Due to the specific nature of the sample, we applied dipolar relaxation enhancement measurements to study the geometry of a protein-protein complex. The paramagnetic centers in question had EPR spectra that were too broad and had such short relaxation time that they could not be studied using the more straightforward PELDOR technique. EPR spectral resolution can be increased appreciably by measuring at a frequency higher than conventional X-band (9 GHz) frequency. The spectra of many paramagnetic species can only be resolved at frequencies higher than 90 GHz. For accurate measurement of the orientation of the vector between two dipolar coupled spins with respect to the g-tensors of the spins, high spectral resolution is required. We therefore performed our EPR measurements at G-band (180 GHz) frequency. Dipolar relaxation measurements were applied to study the complex that is formed by the two electron-transfer proteins cytochrome c and cytochrome c oxidase (CcO) from the soil bacterium Paracoccus denitrificans. We were able to detect dipolar relaxation enhancement due to complex formation of soluble subunit II of P.d. CcO (CcOII) with two substrate cytochromes, which was practically absent in a mixture of CcOII with the negative control protein cytochrome c1. This complex formation was characterized by a pronounced temperature dependence that could be simulated using a home-written computer program. The G-band EPR measurements could not be simulated with a single complex geometry. This provided evidence for the hypothesis that electron-transfer protein complexes are short-lived and highly dynamic; they do not seem to form one specific electron-transfer conformation, but rather move around on each other’s binding surfaces and transfer an electron as soon as the distance between donor and acceptor is short enough. As a test of our simulation program, we also applied dipolar relaxation measurements to specially synthesized organic molecules that contained a nitroxide radical and a metal center. The transverse relaxation of Cu2+-OEP-TPA was compared to the relaxation of Ni2+-OEP-TPA at temperatures between 20 and 120 K. In this temperature range, the nitroxide relaxation was enhanced due to the presence of Cu2+, but not by Ni2+. Similarly, relaxation enhancement was found in the nitroxide-Mn2+ pair in Mn2+-terpyridine-TPA with respect to the terpyridine-TPA ligand. Due to the fast T2 relaxation of the nitroxide radical at high temperatures, the measurements were all performed in the low-temperature regime where the T1 relaxation rate of the metal ion was smaller than the dipolar coupling frequency. In this region, no structural information about the molecule can be deduced, since the dipolar relaxation enhancement is only determined by the T1 of the metal ion. The dipolar relaxation measurements we performed at high field indicated a difference in relaxation times between X-band and G-band frequencies. Extensive T1 - measurements of different paramagnetic centers (CuA, Cu2+) confirmed a strong dependence of T1 on magnetic field in the temperature range where the direct process is the dominating T1 relaxation process. This dependence is very strong (factor of 103 with respect to X-band), but does not follow the B04 dependence predicted in literature. The T1 relaxation of low-spin iron in cytochrome c at high magnetic field, estimated from dipolar relaxation data, is also in agreement with a larger contribution by the direct process (factor of 104). Dipolar relaxation enhancement was found to be a technique that is useful for measuring distances between paramagnetic centers, but only for systems where several important conditions are met, such as: the system exists in one certain static geometry, and the relaxation rate of the fast-relaxing spin is faster than the dipolar coupling frequency within the accessible temperature range. Additionally, it is a great advantage for the analysis of dipolar relaxation data if the procedure of dividing the relaxation trace of the dipolar-coupled slow-relaxing spin by the relaxation trace of the slow-relaxing spin in absence of dipolar coupling can be applied. Another useful application of dipolar relaxation enhancement measurements is the measurement of T1 relaxation of extremely fast-relaxing spins, or spins that are otherwise difficult to detect.
Background Olfactory receptors work at the interface between the chemical world of volatile molecules and the perception of scent in the brain. Their main purpose is to translate chemical space into information that can be processed by neural circuits. Assuming that these receptors have evolved to cope with this task, the analysis of their coding strategy promises to yield valuable insight in how to encode chemical information in an efficient way. Results We mimicked olfactory coding by modeling responses of primary olfactory neurons to small molecules using a large set of physicochemical molecular descriptors and artificial neural networks. We then tested these models by recording in vivo receptor neuron responses to a new set of odorants and successfully predicted the responses of five out of seven receptor neurons. Correlation coefficients ranged from 0.66 to 0.85, demonstrating the applicability of our approach for the analysis of olfactory receptor activation data. The molecular descriptors that are best-suited for response prediction vary for different receptor neurons, implying that each receptor neuron detects a different aspect of chemical space. Finally, we demonstrate that receptor responses themselves can be used as descriptors in a predictive model of neuron activation. Conclusions The chemical meaning of molecular descriptors helps understand structure-response relationships for olfactory receptors and their 'receptive fields'. Moreover, it is possible to predict receptor neuron activation from chemical structure using machine-learning techniques, although this is still complicated by a lack of training data.