Outer membrane protein G (OmpG) is a β-barrel porin that intrinsically generates an ionic current that rapidly fluctuates between open and closed states. These rapid current flucutations are termed gating and are due, in large part, to the movement of loop 6 (L6). It was previously shown that OmpG gating is perturbed by pH where low pH pushes it towards the closed state while the equilibrium is shifted to the open state by increasing the pH. Previous reports showed that two Histidine residues 231 and 261 have a small role in this pH response. However, when we mutate both HIS residues the mutant's gating still remains pH responsive. Therefore the two HIS are not the major residues mediating pH-gating. To reveal the molecular interactions of OmpG dynamics, we used computational modeling to direct our search. We modeled OmpG open and closed states and our results revealed several positive residues (ARG, LYS) in the lumen that potentially stabilized the closed state. We generated SER mutants and found that reducing the positive charge in the lumen shifted the OmpG to a more open state. Of note, the charge on ARG and LYS residues remains unaffected by the pH range 5-7. Thus, we hypothesized that OmpG's pH sensitivity is mediated by residues whose charge is strongly affected by this pH range. Our computational modeling revealed several residues that could be protonated/deprotonated at pH 5-7. These residues evidently included HIS residues but GLU and ASP residues were also revealed as strongly influenced by pH. Interestingly, we found a cluster of GLU residues in the OmpG lumen and mutated them to SER. Removal of this negative cluster pushed the gating towards the closed state. Altering the pH did little to weaken the closed conformation. Therefore our data reveal new molecular interactions between loop 6 and charged clusters found in the lumen and helps to improve our understanding of the intrinsic gating of OmpG.

Computationally-aided revelation of the counteracting forces mediating OmpG loop dynamics

254th National Meeting (2017)

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