Biochemie und Chemie
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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.
Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins.
Background: Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. The higher spatial resolution of super-resolution microscopy techniques demands for precise correction of drift, spectral and spatial offset of images recorded at different axial planes.
Methods: We employ a hydrophilic gel matrix for super-resolution microscopy of cellular structures. The matrix allows distributing fiducial markers in 3D, and using these for drift correction and multi-channel registration. We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores at different axial planes. We calculate a correction matrix for each spectral channel, correct for drift, spectral and spatial offset in 3D.
Results and discussion: We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores in a hydrophilic gel matrix. We distribute multi-color fiducial markers in the gel matrix and correct for drift and register multiple imaging channels. We perform two-color super-resolution imaging of click-labeled DNA and histone H2B in different axial planes, and demonstrate the quality of drift correction and channel registration quantitatively. This approach delivers robust microscopic data which is a prerequisite for data interpretation.
Etablierung, Charakterisierung und Anwendung eines In-vitro-Zellkulturmodells der Blut-Hirn-Schranke
(2001)
Es wurde durch die Optimierung der Isolierung und Kultivierung von cerebralen mikrovaskulären Endothelzellen (BCEC) ein in vitro-Zellkulturmodell der Blut-Hirn-Schranke (BHS) etabliert, das bezüglich seiner Permeabilitäts-Charakteristiken spezifische und herausragende Eigenschaften aufwies. Nach Abschluss der Etablierungs- und Optimierungsphase konnten im bovinen System wiederholt und reproduzierbar transendotheliale elektrische Widerstände (transendothelial electrical resistance, TEER) von zum Teil deutlich oberhalb 1000 Ohm mal cm hoch 2 erzielt werden, und die parazelluläre Permeabilität der bovinen BCEC-Monolayer für Sucrose lag annähernd auf dem niedrigen Niveau hochimpermeabler MDCK-Epithelzell-Monolayer und der entsprechenden in vivo-Parameter. Die per se niedrige Permeabilität des in vitro-BHS-Zellkulturmodells konnte sowohl durch Erhöhung des intrazellulären cAMP-Spiegels der BCEC als auch durch Kokultur der BCEC-Monolayer mit C6-Glioma- Zellen zusätzlich deutlich verringert werden. In Kombination konnte dabei ein TEER von über 3000 Ohm mal cm hoch 2 erzielt werden, einem Wert, der deutlich über dem TEER cerebraler pialer Kapillaren in vivo (1000-2000 Ohm mal cm hoch 2) liegt und in BHS-Zellkultursystemen bisher nicht einmal annähernd erreicht wurde. Der Vergleich des im Rahmen dieser Arbeit etablierten in vitro-BHS-Modells mit gut etablierten und charakterisierten BHS-Modellen bestätigten eindeutig dessen hohe Qualität und herausragende Eigenschaften. Im Rahmen eines indirekt-Kontakt-Kokultursystems konnte in zahlreichen Experimenten eindeutig und reproduzierbar gezeigt werden, dass eine Kokultur von postkonfluenten bovinen oder humanen BCECMonolayern mit humanen Makrophagen zu einer durch deutliche Erhöhung des TEER nachweisbaren Verringerung ihrer Permeabilität führte. Die bei den BCEC durch die Makrophagen induzierte TEER-Erhöhung war mit der durch die Kokultur mit C6-Glioma-ZeIlen bewirkten vergleichbar. Eine proinflammatorische Stimulation der Makrophagen vor Beginn der Kokultur zeigte keine eindeutige Beeinflussung ihrer Wirkung auf die mit ihnen kokultivierten BCEC-Monolayer. Eine Infektion der Makrophagen mit zwei verschiedenen HIV-1-Isolaten zeigte trotz Vorhandenseins einer produktiven HIV-1-lnfektion keine Auswirkung auf die von ihnen bewirkte TEER-Erhöhung bei den kokultivierten BCEC-Monolayer, es konnten diesbezüglich keine Unterschiede zwischen uninfizierten und infizierten Makrophagen festgestellt werden. Eine Erhöhung des intrazellulären cAMP-Spiegels führte bei den mit Makrophagen kokultivierten BCECMonolayern wie bei den mit C6-Glioma-Zellen kokultivierten BCEC-Monolayern zu einer zusätzlichen Steigerung des TEER. Die Inhibition der Adenylat-Cyclase und verschiedener Protein-Kinasen (Protein-Kinasen A, C, G und MLCK) zeigte keine Auswirkung auf die von Makrophagen bewirkte TEER-Erhöhung bei den kokultivierten BCEC-Monolayern; die Aktivierung der Protein-Kinase C führte zu dem gleichen Ergebnis. Die Inhibition der Phospholipase C und der Ca2~-vermittelten Aktivierung des Calmodulins zeigte ebenfalls keine Auswirkung auf die durch die Makrophagen- beziehungsweise C6-Kokultur bewirkte TEER-Erhöhung bei den BCEC-Monolayern. Eine Beteiligung der untersuchten Signaltransduktionswege an dem von Makrophagen oder 06 ausgeübten Einfluss auf die BCEC erscheint somit unwahrscheinlich. Der Befund, dass hämatogene Makrophagen in der Lage sind, bei kultivierten BCEC BHS-spezifische Eigenschaften zu induzieren und/oder aufrechtzuerhalten, ist überaus bemerkenswert, da diese Wirkung bislang ausnahmslos Zellen neuroektodermalen Ursprungs (beispielsweise Astrocyten) sowie glialen Zelllinien zugeschrieben wurde. Hämatogene Makrophagen oder davon abgeleitete Zellen des Hirnparenchyms könnten damit ein Teil der Mikroumgebung sein, die in vivo die charakteristischen Eigenschaften von Hirnendothelzellen modulieren, beispielsweise in Form der perivaskulären Mikrogliazellen, die in engem Kontakt mit den cerebralen Blutgefäßen stehen und höchstwahrscheinlich durch aus dem Blutstrom in das Gehirn einwandernde Monocyten/Makrophagen kontinuierlich ersetzt werden. Das in vitro-BHS-Zellkulturmodell wurde weiterhin für pharmakologische Studien eingesetzt, um den Einfluss von Nanopartikeln, die als potentielle brain targeting-Trägersysteme für Arzneistoffe diskutiert werden, auf die Barriere-Eigenschaften postkonfluenter cerebraler Endothelzellen zu untersuchen. Dabei zeigte sich, dass die Zugabe verschiedener Nanopartikel-Präparationen zu postkonfluenten BCECMonolayern zu einer deutlichen und konzentrationsabhängigen Erhöhung deren Permeabilität für Elektrolyte und makromolekulare Substanzen führte.
The isobaric melting and boiling diagrams for the systems: pyridine/methyltrichlorosilane and pyridine/1,1,1-trichloroethane are reproduced. The existence of the congruently melting addition compound CH3SiCl3· (Pyridin)2 could be confirmed. Some measurements of the molar volume of mixtures between pyridine and methyltrichlorosilane and pyridine and 1,1,1-trichloroethane, respectively, are reported. For both systems the molar excess volume and for the system pyridine/methyltrichlorosilane the molar excess enthalpie have been calculated as a function of the mole fractions.
The isobaric melting and boiling diagrams for the systems: dimethyldichlorosilane/pyridine and 2,2-dichloropropane/pyridine are reproduced. The existence of the incongruently melting addition compounds (CH3)2SiCl2 · (Pyridine)2 and [(CH3)2CCl2]3 · Pyridine could be proved. Some measurements of the molar volume of mixtures of pyridine and dimethyldichlorosilane, and pyridine and 2,2-dichloropropane are reported. For both systems the molar excess volume has been calculated as a function of the mole fractions.
The isobaric melting and boiling diagrams for the systems: trimethylchlorosilane/pyridine and trimethylchloromethane/pyridine are reproduced. Some measurements of the molar volume of mixtures between trimethylchlorosilane and pyridine and trimethylchloromethane and pyridine are reported. For both systems the molar excess volume has been calculated as a function of the mole fractions
Site-specific cleavage of RNAs derived from the PIM1 3′-UTR by a metal-free artificial ribonuclease
(2019)
Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.
The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage.