Mechanistic insight into gramicidin-based detection of protein–ligand interactions viasensitized photoinactivation

Abstract

Among the many challenges for the development of ion channel-based sensorsis the poor understanding of how to engineer modified transmembrane poreswith tailored functionality that can respond to external stimuli. Here, we use themethod of sensitized photoinactivation of gramicidin A (gA) channels in planarbilayer lipid membranes to help elucidate the underlying mechanistic details forchanges in macroscopic transmembrane ionic current observed upon interaction ofC-terminally attached gA ligands with specific proteins in solution. Three differentsystems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95protein (belonging to the ‘PDZ domain-containing protein’) and a gA analogcarrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) ananti-biotin antibody and gA-biotin. The results challenge a previously proposedmechanistic hypothesis suggesting that protein-induced current suppression isdue to steric blockage of the ion passage through gA channels, while they revealnew insight for consideration in alternative mechanistic models. Additionally, wedemonstrate that the length of a linker between the ligand and the gA channelmay be less important for gramicidin-based detection of monovalent compared tomultivalent protein–ligand interactions. These studies collectively shed new light onthe mechanism of protein-induced current alterations in bilayer recordings of gAderivatives, which may be important in the design of new gramicidin-based sensors.