A pH-independent quiet OmpG pore with enhanced electrostatic repulsion among the extracellular loops.

Membrane protein pores have emerged as powerful nanopore sensors for single-molecule detection. OmpG, a monomeric nanopore, is comprised of fourteen β-strands connected by seven flexible extracellular loops. The OmpG nanopore exhibits pH-dependent gating as revealed by planar lipid bilayer studies. Current evidence strongly suggests that the dynamic movement of loop 6 is responsible for the gating mechanism. In this work, we have shown that enhancing the electrostatic repulsion forces between extracellular loops suppressed the pH-dependent gating. Our mutant containing additional negative charges in loop 6 and loop 1 exhibited minimal spontaneous gating and reduced sensitivity to pH changes compared to the wild type OmpG. These results provide new evidence to support the mechanism of OmpG gating controlled by the complex electrostatic network around the gating loop 6. The pH-independent quiet OmpG pores could potentially be used as a sensing platform that operates at a broad range of pH conditions.