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11	Mechanistic Insight into Subunit Stoichiometry for KIR Channel Gating: Ligand Binding, Gating, Binding-Gating Coupling, Coordination, and Cooperativity Wang, Runping 12 January 2007 (has links) Ligand-gated ion channels couple intra- and extracellular chemical signals to cellular excitability. In response to a specific ligand, these channels change their permeability to certain ions by opening or closing their ion conductive pathway, a controlling mechanism known as channel gating. Although recent studies with X-ray crystallography and site-directed mutagenesis have revealed several structures potentially important for channel gating, the gating mechanism is still elusive. Ligand-dependent channel gating involves a series of transient events and asymmetric movements of individual subunits. Understanding of these events appears to be a challenge to current approaches in gating studies by using the homomeric wild-type or mutant channels. I therefore took an alternative approach by constructing heteromeric channels. Subunit stoichiometric studies of the Kir1.1 channel showed that a minimum of one functional subunit was required for the pH-dependent gating of the channel. Four subunits in this channel were coordinated as dynamic functional dimers. In Kir6.2 channel, stoichiometry for proton-binding was almost identical to that for channel gating in the M2 helix, suggesting a one-to-one direct coupling of proton binding in C-terminus to channel gating in M2 helix. Positive cooperativity was suggested among subunits in both the proton binding and channel gating. Ligand binding can be differentiated from channel gating by studying the ATP-dependent gating of Kir6.2 channel. Disruptions in ATP binding were found to change both the potency and efficacy of the concentration-dependent curves, while the baseline activity instead of maximum inhibition was affected by disruptions of channel gating. Four subunits in the Kir6.2 channel undergo negative cooperativity in ATP binding and positive cooperativity in channel gating. The ligand binding was coupled to the gating mechanism in the same subunit and neighboring subunits, although the intrasubunit coupling was more effective. These results are well described with the operational model which we have applied to ion channel studies for the first time. By manipulating the relative distance and the interaction of two transmembrane helices, the inner helix bundle of crossing was found to not only serve as a gate but also determine the consequence of ligand binding. ion channel subunit Stoichiometry ligand binding channel gating binding-gating coupling subunit coordination subunit cooperativity Kir1.1 Kir6.2 pH dependent gating ATP dependent gating. Biology, general
12	Insights into the ATP-dependent reductive activation of the Corrinoid/Iron-Sulfur Protein of Carboxydothermus hydrogenoformans Hennig, Sandra Elisabeth 19 June 2014 (has links) Die Verknüpfung einer exergonischen mit einer endergonischen Reaktion zur Ermöglichung der letzteren ist eine in biologischen Systemen weit verbreitete Strategie. Energetisch benachteiligte Elektronenübertragungsreaktionen im Rahmen der reduktiven Aktivierung von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen, der zu diesen verwandten Benzoyl-CoA-Reduktasen und diversen Cobalamin-abhängigen Methyltransferasen sind gekoppelt an die Hydrolyse von ATP. Der Methylgruppentransfer des reduktiven Acetyl-CoA-Weges von Carboxydothermus hydrogenoformans erfordert den Co(I)-Zustand des Corrinoid/Eisen-Schwefel Proteins (CoFeSP). Um diese superreduzierte Form nach einer oxidativen Inaktivierung zu regenerieren ist ein „Reparaturmechanismus“ erforderlich. Ein offenes Leseraster (orf7), welches möglicherweise für eine reduktive Aktivase von Corrinoid Enzymen (RACE) kodiert, wurde in dem Gencluster der am reduktiven Acetyl-CoA-Weg beteiligten Proteine entdeckt. Im Rahmen dieser Arbeit wurde dieses potenzielle RACE Protein biochemisch und strukturell charakterisiert und die ATP-abhängige reduktive Aktivierung von CoFeSP untersucht. Auf Grundlage der in dieser Arbeit gewonnenen Ergebnisse wurde ein Mechanismus für die ATP-abhängige Aktivierung entworfen. Dieser gibt Einblicke wie die durch ATP-Hydrolyse bereitgestellte Energie einen energetisch ungünstigen Elektronentransfer ermöglichen kann. Hierzu kombiniert RACo das Ausgleichen von Bindungsenergien mit Modulationen am Elektronenakzeptor. Eine vergleichbare Strategie wurde bisher in keinem anderen ATP-abhängigen Elektronenübertragungssystem wie dem von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen oder Benzoyl-CoA-Reduktasen beobachtet und könnte ein für RACE Proteine allgemein gültige Eigenschaft darstellen. / The principle of coupling an exergonic to an endergonic reaction to enable the latter is a widespread strategy in biological systems. Unfavoured electron transfer reactions in the reductive activation of nitrogenases, radical-dependent β,α-dehydratases and the related benzoyl- CoA reductases, as well as different cobalamin-dependent methyltransferases are coupled to the hydrolysis of ATP. The reductive acetyl-CoA pathway of Carboxydothermus hydrogenoformans relies on the superreduced Co(I)-state of the corrinoid/iron-sulfur protein (CoFeSP) that requires a “repair mechanism” in case of incidental oxidation. An open reading frame (orf7) coding for a putative reductive activase of corrinoid enzymes (RACE) was discovered in the gene cluster of proteins involved in the reductive acetyl-CoA pathway. In this work, this putative RACE protein was biochemically and structurally characterised and the ATP-dependent reductive activation of CoFeSP was investigated. Based on the results of this study, a mechanism for the ATP-dependent reactivation of CoFeSP was deduced providing insights into how the energy provided by ATP could trigger this unfavourable electron transfer. The reductive activator of CoFeSP combines balance of binding energies and modulations of the electron acceptor to promote the uphill electron transfer to CoFeSP. A comparable strategy has not been observed in other ATP-dependent electron transfer systems like nitrogenases, radical-dependent β,α-dehydratases and benzoyl- CoA reductases and could be a universal feature of RACE proteins. Bioenergetik Energietransduktion ATP-abhängige Elektronenübertragung ATP-abhängige reduktive Aktivierung B12-abhängige Methyltransferasen Corrinoid/Eisen-Schwefel Protein RACE-Proteine Bioenergetics energy transduction ATP-dependent electron transfer ATP-dependent reductive activation B12-dependent Methyltransferases corrinoid/iron-sulfur protein RACE proteins 570 Biowissenschaften, Biologie 32 Biologie WF 5750 ddc:570
13	Vývoj myšího modelu pro studium chromatin remodelačního genu Smarca5 (Snf2h) / Generation of the Mouse Model to Delineate Function of Chromatin Remodeling Gene Smarca5 (Snf2h) Turková, Tereza January 2016 (has links) The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...
14	Transcriptional regulation and physiological importance of the kdp-system from the halophilic archaeon Halobacterium salinarum Kixmüller, Dorthe 03 April 2012 (has links) The high affinity, ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum, is highly induced under K+ limitation. In contrast to the well-characterized Kdp system in Escherichia coli, in which the kdpFABC genes are transcriptionally regulated by the sensor kinase/response regulator system KdpD/KdpE, transcriptional regulation of the kdp genes in H. salinarum was unknown due to the absence of halobacterial homologues of KdpD/KdpE. Furthermore, the physiological relevance of the KdpFABC K+ uptake system of H. salinarum was puzzling, since hypersaline habitats usually comprise K+ concentrations which do not induce kdp expression. In order to analyze the regulation of kdp gene expression, it was essential to gain information about the transcriptional unit(s) involved. Northern blotting, primer extension analysis and real-time RT-PCR revealed the presence of a polycistronic leaderless kdpFABCQ transcript with a putative kdp terminator or at least a potential mRNA processing site downstream of kdpQ. Furthermore, promoter truncation studies verified the so far only predicted basal transcription elements together with an upstream-located operator sequence. Since deletions of this putative operator sequence did not lead to a constitutive expression, a further component has to be involved in the regulation of the kdpFABCQ genes. However, truncation and scanning mutagenesis analyses of the kdp promoter as well as translational fusions of a halophilic beta-galactosidase to the kdp promoter excluded an additional regulatory element up- or downstream of the basal transcription elements and in the kdp-coding region. These results lead to speculations of multiple basal transcription factors to be involved. Furthermore, an inducible expression vector (shuttle vector) was constructed based on the promoter of the kdpFABCQ operon due to its, K+-sensitive features. Inducible expression systems are yet not available for H. salinarum. The resulting, replicating vector pKIX is functional and enables a K+-dependent expression from the kdp promoter with rather high induction ratios of 50-fold. Expression levels could further be improved by plasmid- and additional chromosomally encoded kdpQ and mutations generated in the kdp promoter. Since transcript levels from pKIX were found to be independent of differential target genes, the general application of pKIX as an inducible expression system is strongly supported and pKIX could, thus, be made accessible to the scientific community. To decipher the physiological relevance of the halobacterial Kdp system, H. salinarum was encountered to desiccation stress and salt crystal (halite) entombment. Halite crystals grown under non-inducing K+ concentrations with entombed strains of H. salinarum and H. salinarum deleted in the kdpFABCQ genes revealed a significantly reduced survival rate of the deletion strain upon recultivation. Additionally, a kdpFABCQ-inducing desiccation stress could already be determined on agar plates under non-limiting K+ concentrations. Furthermore, the cell morphology of H. salinarum entrapped in halite crystals resembled that of H. salinarum grown under K+-limiting conditions. Therefore, the Kdp system promotes survival of H. salinarum under desiccation stress. Furthermore, the Kdp system could be identified as at least one of the systems important for long-term survival of H. salinarum in halite. Halobacterium Halobacterium salinarum H. salinarum halophile halophil archaea archaeon kdp kdpFABC kdpFABCQ kdp-System ATP Kalium Kaliumaufnahme K+ Halit Salzkristall Austrocknung Trockenstress transkriptionelle Regulation Genexpression pKIX induzierbarer Expressionsvektor Expressionsvektor kdpQ Kdp Komplex halobacterial halophilic kdpFABC genes kdp-system high-affinity ATP-dependent potassium potassium pump K+ uptake desiccation desiccation stress halite salt crystal long-term survival transcriptional regulation gene expression inducible expression vector expression vector Kdp complex ddc:570

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