Peuker, Sebastian

English

Adenosin-3´,5´-zyklisches Monophosphat (cAMP) ist ein wichtiger sekundärer Botenstoff, der die Aktivität vieler Proteine reguliert. Eine Gruppe dieser Proteine besitzt eine konservierte Bindestelle für zyklische Nukleotide (CNBD). Ionenkanäle (HCN/CNG), Nukleotidaustauschfaktoren (EPACs), Proteinkinasen (PKA/PKG) und Transkriptionsfaktoren (CAP) werden durch diese CNBD reguliert. Ich habe die Dynamik der CNBD, am Beispiel der isolierten CNBD des zyklisch Nukleotid-gesteuerten Ionenkanals (mlCNGKanals) aus dem Bakterium Mezorhizobium loti, untersucht. Zunächst habe ich die Kinetik der Ligandenbindung mit der „stopped flow“-Technik bzw. der Blitzlichtphotolyse des neuen caged compound BCMACM-8-NBD-cAMP gemessen. Es zeigte sich, dass die Assoziationsraten nicht diffusionslimitiert sind und für die beiden Liganden cAMP und 8-NBD-cAMP bei circa 107 M-1 s -1 liegen. Die R348A-Mutante der CNBD besitzt eine ungefähr 350-fach niedrigere Affinität für 8-NBD-cAMP, was interessanterweise ausschließlich auf die Dissoziationsrate zurückzuführen ist; die Assoziationsrate bleibt durch den Aminosäureaustausch unverändert. Gemeinsam mit neusten Erkenntnissen aus NMR-spektroskopischen Untersuchungen der CNBD deutet das an, dass die ligandeninduzierte Strukturänderung einem „induced fit“-Mechanismus folgt, d. h. die Strukturänderung eine Folge der Ligandenbindung ist. Die Dynamik der CNBD, insbesondere der Sekundärstrukturelemente, wollte ich durch zeitaufgelöste Abstandsänderungen messen. Intramolekulare Abstände ungepaarter Elektronen können mit Hilfe der „double electron-electron resonance“-Methode bestimmt werden. Um möglichst viele strukturelle Zwischenstufen auf dem Weg von der ligandenfreien zur -gebundenen CNBD untersuchen zu können, sollte die CNBD zu verschiedenen Zeitpunkten nachdem sie mit Ligand gemischt wurde, eingefroren werden. Dafür habe ich eine „microsecond freeze-hyper quenching“-Anlage mit einem dynamischen Bereich von 80 µs bis etwa 20 ms gebaut. Ungepaarte Elektronen sollten durch site-directed spin labeling in das Protein eingebaut werden. Das habe ich sowohl mit Hilfe der unnatürlichen Aminosäure p-Acetylphenylalanin als auch über Cysteine versucht. Mit keiner der beiden Strategien konnte eine ausreichende Menge markierten Proteins präpariert werden. Durch eine Verbesserung des Markierungsprotokolls sollte es jedoch möglich sein, ausreichende Mengen markierten Proteins herzustellen und die Dynamik der CNBD zu messen.A number of different proteins are regulated by the second messenger 3'-5'-cyclic adenosine monophosphate (cAMP). Some of these proteins harbor a conserved cyclic nucleotide-binding domain (CNBD). Ion channels (CNG/HCN), nucleotide-exchange factors (EPACs), protein kinases (PKA/PKG), and transcription factors (CAP) are regulated by a CNBD in their activity. My aim was to study the dynamics of the CNBD of a cyclic nucleotide-gated ion channel from the bacterium Mezorhizobium loti (mlCNG-channel). First, I have investigated the kinetics of ligand binding using the stopped-flow technique as well as flash photolysis of the novel caged compound BCMACM-8-NBD-cAMP. I can show that the on-rate of ligand binding for cAMP and 8-NBD-cAMP is lower than the diffusion limit. The low affinity mutant R348A is characterized by a 350-fold lower affinity towards 8-NBD-cAMP compared to the wild-type CNBD. In kinetic terms this is due to an increased off-rate of the mutant, whereas the on-rate is similar to that of the wild-type. Taken together with recent results from NMR spectroscopic studies of the CNBD, I conclude that structural changes induced by ligand binding do follow the induced fit mechanism, i. e. structural changes are a direct consequence of ligand binding. However, to get a more detailed picture of the dynamics of secondary structure rearrangements, I wanted to measure distance changes within the CNBD in a time-resolved manner. Intramolecular distances of unpaired electrons are eventually measured by the double electron-electron resonance (DEER) method. Even though the DEER method is a very slow technique it can be applied to study fast relaxation process, if interstages are trapped. To accomplish this I have built a microsecond freeze-hyper quenching device capable of freeze quenching protein samples 80 µs to 20 ms after they have been mixed with a ligand. Unpaired electrons were introduced in the protein by site directed-spin labeling. I have used the unnatural amino acid p-acetylphenylalanin, in addition to the labeling of cysteines. Up to now, only a single interspin distance could be obtained. Nevertheless, some further improvements of the labeling protocols will enable us to study the dynamics of the CNBD comprehensively

Peuker, S.

MPG.PuRe

Untersuchungen zur Dynamik einer Bindestelle für zyklische Nukleotide

Kölner UniversitätsPublikationsServer

Abstract  A number of different proteins are regulated by the second messenger 3'-5'-cyclic adenosine monophosphate (cAMP). Some of these proteins harbor a conserved cyclic nucleotide-binding domain (CNBD). Ion channels (CNG/HCN), nucleotide-exchange factors (EPACs), protein kinases (PKA/PKG), and transcription factors (CAP) are regulated by a CNBD in their activity. My aim was to study the dynamics of the CNBD of a cyclic nucleotide-gated ion channel from the bacterium Mezorhizobium loti (mlCNG-channel).  First, I have investigated the kinetics of ligand binding using the stopped-flow technique as well as flash photolysis of the novel caged compound BCMACM-8-NBD-cAMP. I can show that the on-rate of ligand binding for cAMP and 8-NBD-cAMP is lower than the diffusion limit. The low affinity mutant R348A is characterized by a 350-fold lower affinity towards 8-NBD-cAMP compared to the wild-type CNBD. In kinetic terms this is due to an increased off-rate of the mutant, whereas the on-rate is similar to that of the wild-type. Taken together with recent results from NMR spectroscopic studies of the CNBD, I conclude that structural changes induced by ligand binding do follow the induced fit mechanism, i. e. structural changes are a direct consequence of ligand binding. However, to get a more detailed picture of the dynamics of secondary structure rearrangements, I wanted to measure distance changes within the CNBD in a time-resolved manner. Intramolecular distances of unpaired electrons are eventually measured by the double electron-electron resonance (DEER) method. Even though the DEER method is a very slow technique it can be applied to study fast relaxation process, if interstages are trapped. To accomplish this I have built a microsecond freeze-hyper quenching device capable of freeze quenching protein samples 80 µs to 20 ms after they have been mixed with a ligand. Unpaired electrons were introduced in the protein by site directed-spin labeling. I have used the unnatural amino acid p-acetylphenylalanin, in addition to the labeling of cysteines. Up to now, only a single interspin distance could be obtained. Nevertheless, some further improvements of the labeling protocols will enable us to study the dynamics of the CNBD comprehensively.  

Untersuchungen zur Dynamik einer Bindestelle für zyklische Nukleotide

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MPG.PuRe

Kölner UniversitätsPublikationsServer