Chemische Modifikation von OmpG und VDAC zur Herstellung von Hybridionenkanälen

Membranproteine wie passive Kanäle und Poren, aktive Transporter und Rezeptoren sind essentiell für die Kommunikation zwischen Zellen, als auch für den Fluss von Substanzen über die von der Zellmembran geformten Permeabilitäts-Barrieren. Aufgrund dieser Eigenschaften stehen Memebranproteine als Angr...

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Bibliographic Details
Main Author: Hirsch, Boris
Contributors: Koert, Ulrich (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2015
Online Access:PDF Full Text
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Membrane proteins like passive channels and pores, active transporters and receptors are essential for the communication between cells as well as for the transport of matter across the permeability barrier formed by cell membranes. Due to this properties, membrane proteins are of interest as targets for the pharmaceutical industry. The performance of ion channels can be modulated with the help of chemical as well as biological functionalization methods, through which insight can be gained into the functionality of these pores. This information could be further used as basis for the design of complex biohybrid systems, for the detection of new targets of drug action and for the understanding of complex biochemical cascades. In this work, chemical modulators for the functionalization of the porins OmpG and VDAC were prepared. The integral membrane protein OmpG, which can be found in the outer membrane of the Gram-negative bacterium Escherichia coli, shows a β-barrel fold formed by 14 β stands with an inner lumen of 12 ∙ 15 Å. VDAC occurs mostly in the outer membrane of mitochondria and forms a β-barrel consisting of 19 β-stands. An α-helix, which is essential for the gating behavior, is located inside the pore and restricts the inner lumen to 15 ∙ 26 Å diameter The modification of OmpG should be accomplished by a twofold S-alkylation of cysteines in the inner conductive pathway by haloacteamides. Therefore, two different pathways approaches were used: One method employed, monofunctionalized bipyridine-linkers were produced, which, after introducing into the pore, could form a stable complex with heavy metal ions like copper and hence, block the pore. In a more straight forward way, bishaloacetamides with different backbones were synthesized, which should form directly a molecular bar through the middle of the conductive pathway und reduce the conductivity. Of the synthesized bipyridine-linkers, one could be successfully reacted with OmpG and an X-ray structure of this biohybrid was obtained showing both bipyridine-linker covalently bound to the inside of the pore. ICP measurements of the copper proportion in the dialyzed biohydrid revealed its ability to bind copper while it was below the calculated copper saturation. However, BLM measurements for the identification of conductivity showed no change after the addition of copper(II)sulfat. For the bishaloacetamid route, the focus lay on terphenyl and azacrownether backbones. A water-soluble terphenyl and an azacrown with glycine spacers could both be transformed to bishaloacetamides which were reacted with OmpG. Both reactions showed ambiguous results towards the two point functionalization. The grown protein crystals of the complex could not be processed. For the modification of a VDAC mutant with a partly missing α-helix, a chemical modified peptide should be introduced to the protein by native chemical ligation (NCL). An appropriate peptide thioester consisting of ten amino acids was synthesized by SPPS and by fragment condensation. Only the peptide made by fragment condensation gave the desired C-terminal deprotected peptide in high purity. Reactions to convert this peptide into the ethylthioester needed for the NCL gave a mixture consisting of ethylthioester and methylester, which could not be separated. It was not possible to get a pure thioester to be reacted with VDAC via NCL.