Doctoral Thesis
Refine
Year of publication
Document Type
- Doctoral Thesis (282) (remove)
Language
- English (282) (remove)
Has Fulltext
- yes (282)
Is part of the Bibliography
- no (282)
Keywords
- Membranproteine (6)
- NMR-Spektroskopie (5)
- RNA (4)
- ABC-Transporter (3)
- Biochemie (3)
- Crystallography (3)
- Kristallographie (3)
- Photosynthese (3)
- Proteinfaltung (3)
- Biochemistry (2)
Institute
- Biochemie und Chemie (282) (remove)
The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
The Na+/proline transporter of E. Coli (PutP) is responsible for the uptake of proline which is subsequently used not only as a carbon and nitrogen source and a constituent of proteins but also as a particularly effective osmoprotectant. However, for a long time there was little known about the single steps in the reaction cycle of this transporter and only few details about its structure-function relationship are available. Aim of the present work was to achieve a deeper understanding about the kinetic properties of the Na+/proline transporter and to get insights into the structure-function relationship of the substrate binding. To answer these questions different techniques were used. By using the novel SSM technique combining the preparation of PutP proteoliposomes it was possible to demonstrate for the first time the electrogenic substrate binding to PutP transporter. Due to rapid solution exchange measurements on the SSM it was additionally possible to obtain time resolved information about the kinetic details of the cytoplasmic substrate binding sites which were not available by previous steady state and equilibrium binding measurements. Pre-steady-state charge translocation was observed after rapid addition of one or both of the cosubstrates Na+ and/or proline to the PutP-WT proteoliposomes adsorbed on the SSM. Thereby it was possible to link the observed electrical signals with the binding activity of PutP. The observed Na+ and/or proline induced charge displacement were assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k > 50 s-1 proceeding the rate limiting step of the reaction cycle. Furthermore, based on the kinetic analysis of the electrical signals obtained from the measurements of PutP on SSM, the following characteristics of the substrates binding in PutP were deduced: (1) both Na+ and proline can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails; while at sufficiently high concentrations, each substrate can bind in the absence of the other; (2) substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with Na+ binding and proline binding is of comparable size and independent of the presence of the respective cosubstrate. In addition, it was concluded that Na+ accesses its binding site through a high-field access channel resulting in a charge translocation, whereas the binding of the electroneutral proline induces a conformation alteration involving the displacement of charged amino acid residue(s) of the protein; (3) Na+ and proline binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate; (4) proline binding proceeds in a two step process: low affinity (~ 0.9 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition; (5) membrane impermeable PCMBS inhibits both Na+ and proline binding to the inside-out orientated PutP transporter, indicating that rather than selectively blocking a specific binding site, PCMBS probably locks the enzyme in an inactive state. The possible targets for this SH-reagent are cysteines 281 and 344 located close to the cytoplasmic surface of the protein. Beyond it, transient electrical currents of PutP were also observed on the BLM after rapid addition of proline in the presence of Na+. This was possible by combining the conventional BLM technique with high-speed flash-photolysis of caged-proline. Indeed the signals on the BLM indicate the detection of a different underlying reaction process in comparison to the data achieved by the SSM technique. This has paved the way for supplemental information about the reaction cycle since it was possible to assign the flash-photolysis BLM signals to the proline binding step followed by the internalization of Na+ and proline into the liposome. Thereby it was found, that the presence of Na+ is indispensable and the time constant for the process is ~ 63 ms. Moreover, structure-function information about the Na+ and proline binding sites of PutP was obtained by investigating the functionally important amino acid residues Asp55, Gly63 and Asp187 with site-directed mutagenesis and the combined SSM technique. One finding is that the mutated proteins PutP-D55C and PutP-G63C showed no activity on the SSM. Therefore, it can be assumed that either both Asp55 and Gly63 are crucial for the structure of PutP protein, or they are located at or close to the Na+ and proline binding sites. Furthermore, the results obtained from PutP-D187N and PutP-D187C mutants on SSM suggest that Asp187 of PutP is likely to be involved in the Na+ binding at the cytoplasmic side of the backward running carrier. Taken together the results of the present work have substantially broadened the known picture of the Na+/proline transporter PutP thereby several steps of the reaction cycle were elucidated, and moreover, valuable insights into the structure-function relationship of the transporter have become available.
Unlimited self-renewal is an absolute prerequisite for any malignancy, and is the ultimate arbiter of the continuous growth and metastasis of tumors. It has been suggested that the self-renewal properties of a tumor are exclusively contained within a small population, i.e., the so-called cancer stem cells. Enhanced self-renewal potential plays a pivotal role in the development of leukemia. My data have shown that APL associated translocation products PML/RARalpha and PLZF/RARalpha increased the replating efficiency of mouse lin-/Sca1+ hematopoietic stem cells (HSCs). This effect is partly mediated by induction of gamma–catenin which is an important mediator of the Wnt signaling pathway and has been shown to be up regulated by the AML associated translocation products(AATPs). Suppression of gamma–catenin by siRNA can abrogate the increased replating efficiency induced by AATPs. Transduction of gamma–catenin in lin-/Sca1+ HSCs led to increased replating efficiency and the expression of stem cell markers Sca1 and c-kit. Additionally it induced accelerated cell cycle progression of mouse bone marrow HSCs. Transduction/transplantation mouse models have shown that ectopic expression of gamma–catenin in HSCs led to acute myeloid leukemia without maturation. These data suggest important roles of Wnt signaling pathway in the leukemogenesis induced by PML/RARalpha, PLZF/RARalpha and AML1/ETO. In contrast to AATPs, CML and Ph+-ALL associated translocation products p185(BCR-ABL) and p210(BCR-ABL) did not affect the self-renewal potential of hematopoietic stem/progenitor cells. However my studies indicated that their reciprocal translocation products p40(ABL/BCR) and p96(ABL/BCR) actually increased the replating efficiency of hematopoietic stem/progenitor cells. The effect is stronger when induced by p96(ABL/BCR) than by p40(ABL/BCR). It is very intriguing that p96(ABL/BCR) can activate Wnt signaling and up regulate the expression of HoxB4. Transduction/transplantation mouse model has shown that p40(ABL/BCR) and p96(ABL/BCR) both have their own leukemogenic potential. Given the fact that leukemic stem cells maintain the growth of tumor and are the origin of relapse, the cure of leukemia is dependent on the eradication of the leukemic stem cell and abrogation of aberrantly regulated self-renewal capability. Both t-RA and As2O3 have been shown to induce complete remission in APL patients with PML/RARalpha translocation product. However, t-RA as a single agent achieves completeremission (CR) but not complete molecular remissions (CMR). Therefore, virtually all patients will experience a relapse within a few months. In contrast to t-RA, As2O3 as a single agent is able to induce CR as well as CMR followed by long-term relapse-free survival in about 50% of APL patients even if relapsed after treatment with t-RA-containing chemotherapy regimens. Nothing is known about the mechanisms leading to the complete different clinical outcomes by the two compounds although both have been shown to induce differentiation of blast cells, proliferation arrest, induction of apoptosis and degradation of PML/RARalpha. We investigated the effect of t-RA and arsenic on PML/RARalpha-expressing cell population with stem cell capacity derived from the APL cell line NB4 as well as Sca1+/lin- murine bone marrow cells. We found that t-RA did not reduce the replating efficiency in PML/RARalpha- and PLZF/RARalpha-infected Sca1+/lincells whereas it selected small compact colonies representing very early progenitor cells. T-RA was unable to reduce the capacity to form colony forming units-spleen (CFU-S) of Sca1+/lin-cells expressing PML/RARalpha, additionally t-RA did not impair the capability of engraftment of NB4 cells in NOD/SCID mouse. On the contrary to t-RA, As2O3 abolished the aberrant self-renewal potential of Sca1+/lin- cells expressing PML/RARalpha. As2O3 not only abolished the replating efficiency of PML/RARalpha positive cells but also completely abrogated the ability of PML/RARalpha-positive HSC to produce CFU-S in vivo. On the contrary to As2O3, t-RA increased the absolute cell number and the percentage of cells in the side population with respect to the whole cell population in NB4 cells. Taken together these data suggest that arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha positive leukemic stem cells. My data prove for the first time that there is a direct relationship between the capacity of compounds to effectively target the LSC and their capacity to eradicate the leukemia, and, thereby, to induce complete molecular remission and long-term relapse-free survival. Thus, in order to increase the curative potential of leukemia therapies, future studies need to include the effect of given compounds on the stem cell compartment to determine their ability to eradicate the LSC.
The transporter associated with antigen processing-like (TAPL) acts as a lysosomal ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for solubilization, purification and functional reconstitution of human TAPL was developed. TAPL was expressed in Sf9 insect cells with the baculovirus expression system and solubilized from crude membranes. By intensive screening of detergents, the mild non-ionic detergents digitonin and dodecylmaltoside were found to be ideal for solubilization with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with a yield of 500 micro g/L cell culture and, subsequently, reconstituted into proteoliposomes. The KM(pep) for the peptide RRYCfKSTEL (f refers to fluorescence label) and KM(ATP) were determined to be 10.5 ± 2.3 micro M and 97.6 ± 27.5 micro M, respectively, which are in the same range as the Michaelis-Menten constants determined in the membranes. The peptide transport activity of the reconstituted TAPL strongly depends on the lipid composition. Interestingly, the E. coli lipids are prefered over other tested natural lipids extracts. Moreover, phosphatidylcholine, the most abundant phospholipid in eukaryotic cells influenced TAPL activity in a dose dependent manner. In addition, some negatively charged lipids like DOPA and DOPS increased peptide transport activity with preference for DOPS. However, DOPE or egg PG which are also negatively charged had no effect. It seems not only the charge but also the specific head group of phospholipids that has impact on the function of TAPL. With the help of combinatorial peptide libraries containing D-amino acid residues at defined positions as well as bulky fluorescein labeled peptides, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. The C-terminal position has the strongest selectivity since modification at this position shows strongest impact on peptide transport. Additionally, positions 2 and 3 of the peptide also have weak influence on peptide selectivity. Subsequently, the residue preferences at the key positions were systematically investigated by combinatorial peptide libraries with defined residues at certain positions. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. The residue preferences at the key positions are valid for peptide substrates with different length, indicating a general rule for TAPL selectivity. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones. By size exclusion chromatography (SEC) and blue native PAGE, TAPL purified in the presence of digitonin or dodecylmaltoside had an apparent molecular weight of 200 kDa which is close to the theoretical molecular mass of the TAPL homodimer (172 kDa). The purified and reconstituted TAPL showed specific ATP hydrolysis activity which can be inhibited by orthovanadate. TAPL in proteoliposomes showed 6-fold higher ATP hydrolysis than digitonin solubilized protein, indicating the phospholipids impact on TAPL function. However, no peptide substrate stimulated ATPase activity was observed. For site-specific labeling of TAPL, eight cysteines in each half transporter were replaced by alanine or valine. The TAPL cys-less mutant showed the same peptide transport activity as TAPL wt. Based on the functional TAPL cys-less mutant, seven single cysteine mutants were introduced into strategic positions. All single cysteine mutants in the TMD did not influence peptide transport, whereas the mutant L701C, which is close to the conserved H-loop motif, displayed impaired transport. TAPL orthologs Haf-4 and Haf-9 from Caenorhabditis elegans possess around 40% sequence identities with TAPL and 50% with each other. Both proteins are putative half transporters and reported to be involved in the intestinal granule formation (Bauer, 2006; Kawai et al., 2009). To further understand the physiological functions of these two proteins, they were expressed in Sf9 insect cells. Haf-4 and Haf-9 showed weak but specific ATP- and peptide-dependent peptide transport activity for the given peptide RRYCfKSTEL. Therefore, it was proposed that the physiological roles for Haf-4 and Haf-9 might be related to their peptide transport activity. Besides forming functional homodimeric complex as estimated by the peptide transport activities, both half transporter could also form heteromers which was confirmed by coimmunoprecipitation. However, the heteromers showed decreased transport activity.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
Metabotropic glutamate receptor subtype 7 (mGluR7) belongs to the family of G-protein coupled receptors. mGluR7 is widely distributed in the brain and primarily localized at presynaptic terminals, where it is thought to regulate neurotransmitter release and synaptic plasticity. Studies have shown that the intracellular C-terminal tail of mGluR7 binds a variety of proteins in addition to trimeric G-proteins. These newly identified protein interactions are believed to play a key role in the synaptic targeting and G-protein dependent signaling of mGluR7. Protein interacting with C kinase 1 (PICK1), a PDZ-domain protein, is a strong interaction partner of mGluR7a. In order to investigate the role of PICK1 in the synaptic trafficking and signaling of mGluR7a, a knock-in mouse line in which the interaction of mGluR7a and PICK1 is disrupted was generated. Analysis of the mutant mice by immunocytochemistry and immunoelectron microscopy showed that the synaptic targeting and clustering of mGluR7a was not altered, indicating that PICK1 is not required for mGluR7a receptor membrane trafficking and synaptic localization. However, when the spontaneous synaptic activity of cerebellar granule cell cultures prepared from both wild-type and knock-in mice was monitored, and L-AP4 (400μm) was found to decrease the frequency, but not the amplitude, of spontaneous excitatory currents in wild-type neurons, while no effect of L-AP4 on spontaneous synaptic activity was observed in knock-in neurons. This indicates that PICK1 binding to the C-terminal region of mGluR7a plays an essential role in mGluR7a mediated G-protein signaling. We examined the threshold sensitivity for the convulsant pentetrazole (PTZ) in knock-in mice. It was found that mGluR7a knock-in mice had a greater sensitivity to PTZ than wild-type mice. Moreover, the surface parietal cortex EEG recordings of the mutant mice revealed spontaneous synchronous oscillation, or "spike-and-wave discharges" (SWD), which displayed similar characteristics to absence-like seizures. It was also observed that the knock-in mice responded to pharmacology as human absence epilepsy. These data suggests that the knock-in mice displayed the phenotype of absencelike epilepsy. Furthermore, the behavioral analysis of the mGluR7a knock-in mice showed no deficits in motor coordination, pain sensation, anxiety as well as spatial learning and memory, thus the interaction of mGluR7a and PICK1 appears not to contribute to these physiological processes. Taken together, our data provides evidence for an important role of PICK1 in Gprotein dependent signaling of mGluR7a, whereas PICK1 is not required for synaptic targeting and clustering of mGluR7a. Our results also provide an animal model of absencelike epilepsy generated by disruption of a single mGluR7a-PDZ interaction, thus creating a novel therapeutic target against this neurological disease.
Modern computational molecular quantum chemical studies, such as the present one, typically employ a wide range of theoretical techniques. The latter are often rather complicated and one should not generally expect that an experimental scientist in the area of physical chemistry, a potential reader of this work, should be familiar with all these techniques. To simplify the reading of the Thesis and to make it self-sufficient, it is supplied with an overview of the employed theoretical methodologies (Chapter 1). The overview explains basic quantum-chemical terminology referred to throughout the Thesis, introduces theoretical foundations of the methods and outlines their properties and limitations. In Part 1.1 of Chapter 1, methods for the solution of the molecular Schrödinger equation are introduced, while in the subsequent Parts 1.2 and 1.3 methods for the solution of the electronic Schrödinger equation are presented to find the ground and excited states, respectively. Part 1.4 is dedicated to basis-set effects which are omnipresent in electronic-structure calculations. It contains a number of unusual insights and concepts proposed by the author and, thus, may be insightful also to experts in quantum chemistry.
In Chapter 2, the phenomenon of acetone-water proton exchange catalyzed by tubular as well as amorphous aggregates of calix[4]hydroquinone (CHQ) macromolecules, which has been observed previously in NMR experiments (Ref. D1D), is investigated by means of correlated quantum-chemical methods. The first part of the study (Section 2.3-2.7) considers concerted proton transfer, assisted by several initially neutral OH-groups in the hydrogen-bonded networks of CHQ aggregates. The second part of the study (Section 2.8-2.13) is dedicated to a second mechanism of proton exchange: step-wise proton transfer via formation of ionic intermediates resulting from CHQ pre-dissociation. CHQ application-specific as well as general conclusions, relevant to the main topic of the Thesis (i.e. influence of specific microsolvation on the considered proton transfer processes), are presented in Section 2.14.
The phenomenon of dual fluorescence observed in clusters of methyl 4-N,N-dimethylaminobenzoate ester (DMABME) and two water molecules in the gas phase, is studied in Chapter 3. Experimentally, the dual fluorescence was detected in experiments combining optical and ground-state ion-depletion infrared spectroscopies in ultracold molecular beams (Ref. D2D). In Section 3.3, calculated ground-state infrared spectra are presented that allow to identify the structures of those isomers, which are present in the gas-phase, as well as the structure of the isomer responsible for dual fluorescence. To further understand the reaction mechanism of dual fluorescence, excited-state potential energy surfaces of the identified isomers were computed along the relevant twisted intermolecular charge-transfer formation coordinate and the mechanism of energy dissipation in these complexes was investigated (Section 3.4-3.5) (Ref. D3D). A brief summary of the main results of this chapter and conclusions are given in Section 3.6. Finally, in Section 3.7 a complementary benchmark study of the quality of ground-state potential energy surfaces of prototypical hydrogen-bonded systems (ammonia-water and formic acid-water dimers) obtained at the level of BSSE-corrected MP2 combined with moderate basis sets, has been conducted. The quality of potential energy surfaces was tested with respect to basis-set size, level of electron correlation and anharmonicity effects and the applied methodology to identify the IR spectrum of hydrated DMABME complexes (Section 3.3) has been found to be sufficient to uniquely assign the IR spectra.
Nucleotide-binding domains (NBDs), roughly 27 kDa in size, are conservative components of the large family of ABC (ATP-binding cassette) transporters, which includes importers, exporters, and receptors. NBDs or ABC-ATPases supply energy for the translocation of a vast variety of substrates across biological membranes. Despite their hydrophilic sequence, many NBDs tend to aggregate and precipitate in solution upon isolation from the complete transporter. The conditions stabilizing an extremely labile NBD component of the E.coli HlyA transporter, HlyB-NBD, were developed. As a result, the pure highly concentrated enzyme was protected from precipitation for months that allowed screening of the unlimited crystallization conditions in the presence of different substrates and performance of the reproducible functional assays. HlyB-NBD was characterized in regard to its uncoupled ATPase activity, oligomeric state, and stability in solution. Comparative analysis of protein stability and ATPase activity in various buffers suggested an inverse relationship between the two. Kinetic analysis of ATPase activity revealed ATP-induced protein dimerization. Gel-filtration experiments with the wild type protein and H662A-mutant of HlyB-NBD provided further evidence of protein dimerization in the presence of ATP. The crystal structures in post- and pre-hydrolysis nucleotide-bound states of HlyB-NBD were determined at 1.6Å and 2.5Å resolution, respectively. While the hydrolytically deficient H662A mutant of HlyB-NBD was crystallized as a stable dimer in the presence of ATP or ATP-Mg2+, with two nucleotide molecules sandwiched between the two monomers, the same protein was shown to be a monomer in the ADP-loaded state. The wild type protein failed to develop crystals with bound ATP, yet formed ADP-bound crystals identical to those of the H662A-mutant. The X-ray structures of HlyB-NBD in various states of the hydrolytic cycle and the functional studies of the enzyme have provided an opportunity to characterize enzyme-substrate complexes and protein-protein interactions between the NBD subunits in great detail. Comparison of the nucleotide-free, the ADP-, and the ATP-loaded states revealed oligomeric and conformational changes of the protein upon substrate binding and resulted in a molecular picture of the catalytic cycle. The correlated results of the structural and functional investigations of HlyB-NBD are discussed with relation to the mechanism of action of ABC transporters.
Prostaglandin E2 is the major prostaglandin involved in colorectal carcinogenesis. The biosynthesis of prostaglandin E2 is accomplished by several terminal prostaglandin E synthases through catalytical conversion of the cyclooxygenase product prostaglandin H2. Among the known terminal prostaglandin E synthases, microsomal prostaglandin E synthase type 1 and type 2 were found to be overexpressed in colorectal cancer, however the role and regulation of these enzymes in this tumor entity are yet not fully understood. Here we report that the cyclopentenone prostaglandins 15-deoxy-D12,14-prostaglandin J2 and prostaglandin A2, which have been shown to modulate cell growth and neoplasia, selectively down-regulate microsomal prostaglandin E synthase type 2 mRNA and protein expression in the human colorectal carcinoma cell lines Caco-2 and HCT 116. This effect appeared to be PPARgamma independent and was not found to require G-protein-coupled receptor activation. Instead, inhibition of microsomal prostaglandin E synthase type 2 by cyclopentenone prostaglandins may be mediated by covalent binding of the cyclopentenone ring to cysteine residues on signalling molecules or via a redox-dependent mechanism. Inhibition of microsomal prostaglandin E synthase type 2 was subsequently followed by decreased prostaglandin E synthase activity, which in turn contributed at least in part to the anti-proliferative action of cyclopentenone prostaglandins in HCT 116 cells. Collectively, these data unravel a novel mechanism for the growth-inhibitory effects of cyclopentenone prostaglandins and expose microsomal prostaglandin E synthase type 2 as a new potential target for pharmacological intervention in the treatment of colorectal cancer.