Ion-Channel Engineering with mVDAC1 and OmpG

We explored the potential of Ion-Channel Engineering^[1] using monomeric porins like OmpG (Outer Membrane Protein G) from Escherichia coli^[2] and applying different chemical strategies. Therefrom derived techniques were then transferred to the more challenging target mVDAC1 (murine Voltage Dependent Anion Channel 1), the most abundant protein in the outer mitochondrial membrane mediating metabolite flow and involved in mitochondrial apoptosis pathways.^[3] Besides the implementation of routes for chemical switching the main goal here is to elucidate the gating mechanism of VDACs.

The first chemical strategy utilizes native chemical ligation providing the possibility to incorporate artificial amino acids, the second depends on the specific modification of engineered cysteines with nucleophilic chemicals and disulfides. In a second step primary channel hybrids may be further modified under native conditions with click-chemistry. Purified and refolded biohybrids are characterized by X-ray crystallography and planar lipid bilayer recordings, both benefit from the monomeric nature of the porins examined.^[4,5]

We present data on biohybrids of an enhanced OmpG pore obtained by different strategies. Our findings of an side-wall attached mVDAC1 variant with engineered disulfide bridges severely interfering with the usual gating of the pore, verify the position of the N-terminal α-helix as well as its major involvement in gating.

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