Tuning Protein Discrimination Through Altering the Sampling Interface Formed between the Analyte and the OmpG Nanopore.

Abstract

Nanopore sensors capable of distinguishing homologous protein analytes are highly desirable tools for proteomics research and disease diagnostics. Recently, an engineered outer membrane protein G (OmpG) nanopore with a high-affinity ligand attached to a gating loop 6 showed specificity for distinguishing homologous proteins in complex mixtures. Here, we report the development of OmpG nanopores with the other six loops used as the anchoring point to host an affinity ligand for protein sensing. We investigated how the analyte binding to the affinity ligand located at different loops affects the detection sensitivity, selectivity, and specificity. Our results reveal that analytes weakly attracted to the OmpG nanopore surface are only detectable when the ligand is tethered to loop 6. In contrast, protein analytes that form a strong interaction with the OmpG surface via electrostatic attractions are distinguishable by all seven OmpG nanopore constructs. In addition, the same analyte can generate distinct binding signals with different OmpG nanopore constructs. The ability to exploit all seven OmpG loops will aid the design of a new generation of OmpG sensors with increased sensitivity, selectivity, and specificity for biomarker sensing.