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Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-ß-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-ß-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable ‘base-paired’ geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.
The development of resistance to multiple drugs is a major problem in treatment of number of infectious diseases and cancer. The phenomenon of multidrug resistance (MDR) is based on the synergetic interplay of a number of mechanisms such as target inactivation, target alteration, prevention of drug influx as well as active extrusion of drugs from the cell. The latter is mediated by over-expression of multidrug efflux pumps. The first discovered and the best characterized until now the human MDR transporter is P-glycoprotein. It is a member of the ATP binding cassette (ABC) superfamily and acts as an active transporter for a variety of anticancer agents using the energy released by ATP hydrolysis. The closest structure and functional homologue of P-glycoprotein found in bacteria is LmrA from Lactococcus lactis. The major goals of this work are to establish the selective isotope labelling of LmrA in Lactococcus lactis, to optimize LmrA sample preparation for solid-state NMR, and finally to perform first solidstate NMR investigations on LmrA shedding light on its catalytic cycle and substrate binding. For a long time the solid-state NMR applications to biological science has been limited to investigation of small molecules mostly. Recently, the solid-state NMR methods have shown potential for structuraland non-perturbing, site directed functional studies of large membrane proteins as well as ligands bound to them. However, to our knowledge neither selective isotope amino acid labelling of any ABC transporter, nor NMR investigations on full-length ABC transporter have been reported to date. Solidstate NMR experiments on a membrane protein require reconstitution of purified proteins into a membrane environment at a high density and either isotopic enrichment of the protein or bound drugs or inhibitors. Therefore, the large quantities of LmrA reconstituted at a high density in lipid membranes, sufficient for advanced NMR studies have been produced and its functional state in reconstituted form has been assessed. In the next step, a procedure for cost effective selective amino acids isotope labelling of LmrA in Lactococcus lactis has been established. Using this protocol deuterium alanine labelled LmrA reconstituted into E. coli liposomes has been prepared. Deuterium NMR has been used extensively to assess the proteins dynamics in past. However, it has never been applied to ABC transporter. Here, we report 2H NMR on selective alanine isotope labelled LmrA which has been used to shed light on the dynamics changes in the protein occurred under AMP-PNP, non-hydrolysable ATP analogue, binding and in ATP/ADP-Vanadate trapped state. It has been found that the major conformation changes affecting the protein motional characteristics occur in the ATP binding domains but not in the transmembrane domains. Additionally, the binding of several substrates to LmrA has been studied by fluorescence spectroscopy as well as by 19F and 31P solid-state NMR. The binding constants for several LmrA substrates have been obtained by fitting the concentration dependant tryptophan intrinsic fluorescence quenching curves. Based on the fluorescence studies and solid-state NMR data, the conformation changes in LmrA under substrate binding have been discussed. In addition, the preferable location of nine LmrA and P-glycoprotein substrates within the model membrane has been studied via 1H-MAS-NOESY-NMR. The results have been interpreted with respect to LmrA and P-glycoprotein binding site accessibility from the membrane interface region.
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.
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 ...
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.
The N-terminal domain (matrix protein or MA) of a retroviral Gag polyprotein precursor plays a critical role in several stages of the retrovirus life cycle. MA is involved in the effective membrane targeting, assembly and release of the immature viral particles from the infected cell. In order to understand the structural basis of these functions, the full length MA from Moloney Murine Leukemia Virus (MoMuLV) was purified and the solution structure of the MA MoMuLV was determined by means of heteronuclear high-resolution NMR spectroscopy and compared with that of the X-ray diffraction analysis as well as with the structures of several MA proteins from geterologous viruses. Structural features were also obtained from CD spectroscopy, dynamic light scattering, sedimentation velocity, differential scanning calorimetry and other methods. It was found that the MA MoMuLV globular core (residues 8-98) is comprised of 7 well-defined helices (five alpha-helices and two 310 helices), with the general fold typical for MA proteins from other retroviral species. The N-terminus (residues Met1-Leu7) and the C-terminal proline-rich part (residues Pro103-Tyr131) are not structured in solution. Although MA MoMuLV has a low sequence identity compared with other matrix proteins for which the three-dimensional structure is known, it was shown that its overall topology and pattern of secondary structural units is similar to other retroviral matrix proteins. The monomeric state is observed for the correctly folded MA MoMuLV in a variety of external conditions and protein concentrations, indicating that virion assembly starts with the plasma membrane targeting of the nascent Gag precursor. The denaturation of MA MoMuLV is irreversible and is connected with protein aggregation. For Moloney Murine Leukemia Virus (MoMuLV) a proteolytic processing of the R-peptide (last 16 amino acids from the C-terminus of the Envelope protein (Env)) has been described as a second mode of fusion and activation preceding the receptor contact between the viral particle and the cellular membrane. An interaction between the R-peptide and MA MoMuLV has been proposed, since the R-peptide and MA are localized at the inner part of the membrane. Therefore the interaction between 15N labelled purified MA MoMuLV and synthesized R-peptide has been investigated using high-resolution NMR. It was found that in water solution MA MoMuLV and R-peptide do not form a tight complex, but in a mature virion in the presence of membranes or other protein factors it might be possible. In the case of HIV-1 the cytoplasmic part (EnvC) of the Env protein is much longer than in other retroviruses and again as for MoMuLV little is known about the interaction between EnvC and HIV MA. Hence, the full length HIV MA, and the last 150 amino acids from HIV Env have been subcloned with suitable expression vectors, purified and analysed by native gel electrophoresis, a pull down assay and by high resolution NMR for the purpose to detect the complex formation of EnvC and HIV MA. Finally, after all those experiments, it was found that a stable complex is not formed, but a weak interaction between the two proteins can not be excluded.
Background: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.
One of the central research topics in the field of biophysical chemistry is the structure and function of membrane proteins involved in energy transduction. Both, the aerobic and the anaerobic respiration include electron transfer and proton translocation across the mitochondrial and bacterial membranes. These electron transfer processes lead to changes in oxidation states of cofactors some of which are paramagnetic. Therefore, EPR spectroscopy is the method of choice to obtain electronic and structural information directly related to the function of the respiratory chain proteins. In this work, multifrequency continuous wave (CW) and pulsed EPR spectroscopy has been used to characterize the molybdenum active site of polysulfide reductase (Psr) from the anaerobic bacterium Wolinella succinogenes and the protein-protein complex between cytochrome c oxidase (CcO) and cytochrome c from the aerobic bacterium Paracoccus denitrificans. Molybdenum in Psr-Psr is an enzyme essential for the sulfur respiration of Wolinella succinogenes. Biochemical studies suggested that the active site of this enzyme contains a mononuclear Mo center, which catalyzes the reduction of the substrate polysulfide to sulfide. Until now there is no crystal structure available for Psr. Consequently, current characterizations of this enzyme have to rely on biochemical and spectroscopic investigations. Within the present work, CW and modern pulsed EPR techniques were applied to investigate its catalytically active site. In the first part of this thesis, different redox agents have been used to generate paramagnetic states of Psr. Multifrequency CW-EPR spectroscopy was applied to identify the Mo(V) states. Using simulations of the experimental spectra, three spectroscopically distinct states have been identified based on the Mo hyperfine- and g-tensor values. Comparison of their EPR parameters with those of related enzymes indicated five or six sulfur ligands at the Mo center depending on the state. The state generated by addition of polysulfide is suggested to be the catalytically active form, in which the Mo is coordinated by a sulfur of the polysulfide chain as the sixth ligand. 33S (I = 3/2) labeled polysulfide was prepared to probe the proximity of the polysulfide to the molybdenum center via its hyperfine coupling. 1D-ESEEM and 2D122 HYSCORE spectroscopy was used to detect these hyperfine and quadrupole interactions, which are too small to be observed in conventional CW EPR spectra. To date there has been only one pulsed-EPR study involving a 33S nucleus [Finazzo et.al. 2003]. The reasons are that this nucleus has a high nuclear spin of I = 3/2 and a large nuclear quadrupole moment in addition to the low Larmor frequency. All these make the detection of sulfur and the extraction of structural information demanding. However, analysis of the 2D-data led to a Mo(V) 33S distance in a range of about 2 to 2.5 Å. Mo-S distances found in molybdenum enzymes of the same family are in a range of 1.8 to 2.8 Å suggesting that the 33S is indeed the sixth ligand of the Mo(V) center and demonstrating that polysulfide is the actual substrate for this enzyme. Thus HYSCORE experiments have been proved to be a powerful technique to gain further insight into the active site structures of molybdenum enzymes and the trafficking of substrate atoms during catalysis. Density functional theory (DFT) calculations together with quantitative numerical simulations of the 2D-data will help to obtain more structural details about the molybdenum binding site in Psr. CcO:cytochrome c complex Protein-protein complex formation is an important step in energy conversion biological processes such as respiration and photosynthesis. These protein-protein complexes are involved in long range electron transfer reactions and are known to be of transient nature. Within the bacterial and mitochondrial respiratory electron transport chains such a complex is formed between CcO and cytochrome c. Upon complex formation cytochrome c donates the electrons required for the CcO catalyzed reduction of dioxygen to water. Here, the protein-protein complex formation between CcO and cytochrome c from Paracoccus denitrificans was investigated by pulsed EPR spectroscopy. The idea was to use the relaxation enhancement due to the distance and orientation dependent magnetic dipole-dipole interaction between the paramagnetic centers in the different CcO constructs and cytochromes. Two-pulse electron spin echo experiments were carried out on mixtures of the CuA containing soluble subunit II or the full size CcO with the physiological partner cytochrome c552 or horse heart cytochrome c. Significantly enhanced relaxation of CuA due to specific protein-protein complex formation has been observed in all four cases. In contrast the non-binding cytochrome c1 showed only a very weak relaxation enhancement due to unspecific protein-protein interactions. The echo decays of the slowly relaxing observer spin (CuA of CcO) measured in the absence and presence of the fast relaxing spin (Fe(III) of cytochrome c) permitted the extraction of the pure dipolar relaxation contributions for the different complexes. Measurements at different temperatures proved the dipolar nature of the relaxation enhancement. Furthermore, it was demonstrated experimentally that this approach also works for the full-size CcO, which contains four paramagnetic metal centers, in complex with cytochrome c. Quantitative simulations of the data suggest a broad distribution in distances (2 - 4 nm) and orientations between the CuA and Fe(III) in the complex between CcO and cytochrome c. High-field EPR spectroscopy will be useful to further analyze and prove these complex structures. Within the present work, it has been shown that pulsed relaxation enhancement experiments can be used to investigate the distance and relative orientation between paramagnetic metal centers. Furthermore, it has been demonstrated on a qualitative level, that this method can be used complimentary to other biophysical approaches to study transient electron transfer protein-protein complexes. Finally, within this work it has been proven that this method can be applied also to biological systems where more than two paramagnetic centers are present. This is particularly interesting for supercomplexes between membrane proteins.
G protein-coupled receptors (GPCRs) play regulatory roles in many different physiological processes and they represent one of the most important class of drug targets. However, due to the lack of three-dimensional structures, structure based drug design has not been possible. The major bottleneck in getting three-dimensional crystal structure of GPCRs is to obtain milligram quantities of pure, homogenous and stable protein. Therefore, during my Ph.D. thesis, I focused on expression, characterization and isolation of three GPCRs namely human bradykinin receptor subtype 2 (B2R), human angiotensin II receptor subtype 1 (AT1aR), and human neuromedin U receptor subtype 2 (NmU2R). These receptors were heterologously produced in three different expression systems (i.e. Pichia pastoris, insect cells and mammalian cells), biochemically characterized and subsequently solubilized and purified for structural studies The human bradykinin receptor subtype 2 (B2R) is constitutively expressed in a variety of cells, including endothelial cells, vascular smooth muscle cells and cardiomyocytes. Activation of B2R is important in pathogenesis of inflammation, pain, tissue injury and cardioprotective mechanisms. During this study, recombinant B2R was produced in methylotrophic yeast Pichia pastoris (3.5 pmol/mg), insect cells (10 pmol/mg) and mammalian cells (60 pmol/mg). The recombinant receptor was characterized in terms of [3H] bradykinin binding, G protein coupling, localization, and glycosylation. Subsequently, it was solubilized and purified using affinity chromatography. Homogeneity and stability of purified B2R was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from BHK cells and Sf9 cells, which were used for three-dimensional crystallization attempts. The second receptor, which I worked on, is human angiotensin II receptor subtype 1 (AT1aR). AT1aR is distributed in smooth muscle cells, liver, kidney, heart, lung and testis. Activation of AT1aR is implicated in the regulation of blood pressure, hypertension and cardiovascular diseases. Recombinant AT1aR was produced at high levels in Pichia pastoris (167 pmol/mg), while at moderate levels in insect cells (29 pmol/mg) and mammalian cells (32 pmol/mg). The recombinant receptor was characterized in terms of [3H] angiotensin II binding, localization, and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Homogeneity and stability of purified AT1aR was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from Pichia pastoris, which were used for threedimensional crystallization attempts. In addition to B2R and AT1aR, I also attempted to produce and isolate the human neuromedin U receptor subtype 2 (NmU2R), which was deorphanized recently. It is found in highest abundance in the central nervous system, particularly the medulla oblongata, spinal cord and thalamus. The distribution of this receptor suggests its regulatory role in sensory transmission and modulation. During this study, recombinant NmU2R was produced in Pichia pastoris (6 pmol/mg) and BHK cells (9 pmol/mg). Recombinant receptor was characterized with regard to [125I] NmU binding, localization and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Due to its low expression level, further expression optimization is required in order to obtain milligram amounts for structural studies. The long-term goal of this study was to obtain three-dimensional crystal structure of recombinant GPCRs. However, 3-dimensional crystallization of human recombinant membrane proteins still remains a difficult task. On the other hand, recent advances in the solid-state NMR spectroscopy offer ample opportunities to study receptor-ligand systems, provided milligram quantities of purified receptor are available. Therefore, in parallel to 3-dimensional crystallization trials, purified B2R was also used for solid-state NMR analysis in order to investigate the receptor bound conformation of bradykinin. Preliminary results are promising and indicate significant structural changes in bradykinin upon binding to B2R. Further experiments are ongoing and will hopefully result in the structure of receptor bound bradykinin. One of the challenges in GPCR crystallization is the small hydrophilic surface area that is available to make crystal contacts. One possibility to overcome this problem can be the reconstitution of a GPCR complex with an interacting protein for cocrystallization. For this purpose, I coexpressed B2R and AT1aR, which form a stable heterodimer complex, in BHK cells. I could successfully isolate the heterodimer complex by using two-step affinity purification. Unfortunately, this complex was not stable over time and disassociates within three days of purification. However, during coexpression of B2R and AT1aR in BHK cells, I observed that B2R was localized in the plasma membrane in coexpressing cells while it was retained intracellularly when expressed alone. This coexpression of AT1aR with B2R resulted in a four-fold increase in [3H] bradykinin binding sites on the cell surface. In addition, these two receptors were cointernalized in response to their individual specific ligands. Interestingly, colocalization of B2R and AT1aR was also found in human foreskin fibroblasts (which endogenously express both receptors), in line with the possibility that heterodimerization may be required for surface localization of B2R in native tissues as well. This is the first report where surface localization of a peptide GPCR is triggered by a distantly related peptide GPCR. These data support the hypothesis that heterodimerization may be a prerequisite for cell surface localization of some GPCRs. A second approach that I followed to stabilize the purified B2R was to reconstitute the B2R-β-arrestin complex. β-arrestin is a cytosolic protein that participates in agonist mediated desensitization of GPCRs and therefore dampens the cellular responses initiated by the activation of GPCRs. I tried to reconstitute B2R-β-arrestin complex in vitro by mixing purified B2R and purified β-arrestin. But, no interaction of these two proteins was observed in the pull-down assays. However, a C-terminal mutant of B2R (where a part of the C-terminus of the B2R is exchanged with that of the vasopressin receptor) was found to interact with β-arrestin in vitro as revealed by pull-down assays. In conclusion, this work establishes the production, characterization and isolation of three recombinant human GPCRs. Recombinant receptors were produced in milligram amounts and therefore, pave the way for structural analysis. The heterodimer complex of B2R-AT1aR and B2R-β-arrestin complex can be of great help during crystallization. In addition, it was also found for the first time that the surface localization of a peptide GPCR can be triggered by heterodimerization with a distantly related peptide GPCR.
Background: Particle Swarm Optimization (PSO) is an established method for parameter optimization. It represents a population-based adaptive optimization technique that is influenced by several "strategy parameters". Choosing reasonable parameter values for the PSO is crucial for its convergence behavior, and depends on the optimization task. We present a method for parameter meta-optimization based on PSO and its application to neural network training. The concept of the Optimized Particle Swarm Optimization (OPSO) is to optimize the free parameters of the PSO by having swarms within a swarm. We assessed the performance of the OPSO method on a set of five artificial fitness functions and compared it to the performance of two popular PSO implementations. Results: Our results indicate that PSO performance can be improved if meta-optimized parameter sets are applied. In addition, we could improve optimization speed and quality on the other PSO methods in the majority of our experiments. We applied the OPSO method to neural network training with the aim to build a quantitative model for predicting blood-brain barrier permeation of small organic molecules. On average, training time decreased by a factor of four and two in comparison to the other PSO methods, respectively. By applying the OPSO method, a prediction model showing good correlation with training-, test- and validation data was obtained. Conclusion: Optimizing the free parameters of the PSO method can result in performance gain. The OPSO approach yields parameter combinations improving overall optimization performance. Its conceptual simplicity makes implementing the method a straightforward task.