Ion-Channel Engineering: Das monomere Porin OmpG als Modell

Membrane proteins like passive channels and pores, active transporters and receptors mediate the essential flow of information and matter across the permeability barrier generated by cell membranes. Due to their central role in the cell’s survival, membrane proteins form important targets for drug development by the pharmaceutical industry. Ion-Channel Engineering (ICE) utilizes chemical and biological methodologies to modify naturally occurring channels and pores to access and elucidate their function. Information derived from these membrane proteins can be applied to the design of complex systems as performed in bottom-up approaches of synthetic biology. This work exploits the potential of the monomeric porin OmpG to serve as a model for ICE as OmpG provides unique structural features amongst this class of proteins. This integral membrane protein from the outer membrane of the Gram-negative bacterium Escherichia coli shows a beta-barrel fold formed by 14 beta-strands and shows a large inner lumen of 12 · 15 Å diameter. The porin functions as a monomer and can be either extracted from membrane fractions or refolded from inclusion bodies using the rapid dilution technique. Two approaches were chosen to functionalize the OmpG porin: Functionalization of cysteines in the inner conductive pathway by S-alkylation or by the introduction of chemical alterations by N-peptides using the native chemical ligation (NCL) technique. As second step, copper (I)-catalyzed click chemistry was performed for diversification. Obtained hybrids were characterized by a combined approach using SDS-PAGE, fluorescence spectroscopy, mass spectrometry, black lipid membrane measurements (BLM measurements) and protein crystallography. All approaches yielded functionalized protein hybrids. Additionally, the first X-ray structure of a synthetically modified OmpG pore could be presented. The data including BLM-measurements mostly raised two challenges: reduction of flickering and attachment of the compounds to the pore for mobility reduction. To enforce a stable open state of the pore two deletion variants of the OmpG pore were prepared lacking the flexible loop6. Both variants could be refolded and formed functional channels with improved gating characteristics as indicated by BLM recordings. X-ray crystallographic data could be obtained for the variant bearing the larger deletion. The structural data revealed a novel, triclinic crystal form for the OmpG pore. Analysis of all observed crystal packings identified two hydrophobic interfaces, of which at least one is part of every structural assembly. Two strategies for the advanced attachment of a molecular bar through the middle of the OmpG conductive pathway were examined. One strategy focused on the direct introduction of the bar using the modification of cysteines by bifunctional labeling agents. The other strategy established the bar by later complexation of a metal ion using two independent, cystein-attached bipyridine ligands. The latter method appeared to have succeeded in establishing a molecular bar due to incorporated copper found by ICP-mass spectrometry and planar lipid bilayer recordings. In general, OmpG serves as an ideal template for ICE due to its unique structural features. This membrane protein could be optimized for sensing applications and functionalized through application of diverse methods.