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Elektrophysiologische Charakterisierung des mitochondrialen Porins VDAC1 und des antimikrobiellen Peptids Dermcidin in lösungsmittelfreien Modellmembranen

dc.contributor.advisorSteinem, Claudia Prof.
dc.contributor.authorWeichbrodt, Conradde
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titleElektrophysiologische Charakterisierung des mitochondrialen Porins VDAC1 und des antimikrobiellen Peptids Dermcidin in lösungsmittelfreien Modellmembranende
dc.title.translatedElectrophysilogical characterization of the mitochondrial porin VDAC1 and the antimicrobial peptide Dermcidin in solvent-free model membranesde
dc.contributor.refereeSteinem, Claudia Prof.
dc.description.abstractengSolvent free lipid bilayers were used for the electrophysiological characterization of the most abundant protein of the outer mitochondrial membrane, the isoform 1 of the voltage dependent anion channel (VDAC1), and the antimicrobial peptide Dermcidin. The bilayers were prepared via spreading of giant unilamellar vesicles (GUVs) over a single pore in a borosilicate glass surface. To gain deeper insight in its gating behavior, wild type VDAC1 as well as two VDAC1 mutants were incorporated into GUVs which then were spread over the aperture. Subsequent analysis of single channels at varying transmembrane potentials Um revealed the transition between an open conformation with a conductance of Go = 4.0 nS at low potentials and a closed conformation with Gc = 2.0 nS in 1 M KCl for Um ≥ 30 mV and Um ≤ 30 mV for all VDAC1 variants. This gating between the main VDAC1 states with low transition rates of kmg = 0.1 2 s-1 did not necessarily occur at high transmembrane potentials as the open probability Po did not fall below 90 % even at Um ≥ 30 mV on a single channel level. Furthermore, a VDAC1 mutant with three additional amino acids at its N-terminal end (RGS VDAC1) exhibited a previously not characterized second gating that superimposed the main gating of VDAC1. Closer analysis of this fast gating resulted in a lower amplitude of ΔGfg = 0.5 1.2 nS and much higher transition rates of kfg = 18 911 s 1 with higher rates at low voltages and vice versa. A second mutant (V17C/A205C VDAC1) contained an artificially introduced disulfide bond that prevented the movement of a certain protein structure as putative molecular basis of the VDAC1 conductance transition. However, V17C/A205C VDAC1 did not exhibit an altered behavior compared to the wild type VDAC1. The antimicrobial activity of Dermcidin (DCD) mainly consists of its ability to perforate lipid bilayers. To elucidate the postulated zinc dependency of the DCD activity, stable membranes were prepared and subsequently incubated with DCD in a zinc free environment or in the presence of Zn2+ ions. High DCD activity was detected in the presence of Zn2+ ions whereas no activity was found under zinc free conditions or in the presence of Mg2+ ions. These findings and the inactivation of DCD via the alteration of a zinc binding site in a mutant (H38A DCD) proved the zinc dependency of the DCD
dc.contributor.coRefereeSchroeder, Jörg Prof.
dc.contributor.thirdRefereeDiederichsen, Ulf Prof.
dc.contributor.thirdRefereeFicner, Ralf Prof.
dc.contributor.thirdRefereeTittmann, Kai Prof.
dc.contributor.thirdRefereeMeinecke, Michael
dc.subject.gerantimikrobielles Peptidde
dc.subject.engantimicrobial peptidesde
dc.subject.engmodel membranesde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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