title: Mechanistic insight into gramicidin-based detection of protein-ligand interactions via sensitized photoinactivation.
abstract: Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein-ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia.
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Rokitskaya
authors.forename: Michael X
authors.initials: MX
authors.lastname: Macrae
authors.forename: Steven
authors.initials: S
authors.lastname: Blake
authors.forename: Natalya S
authors.initials: NS
authors.lastname: Egorova
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kotova
authors.forename: Jerry
authors.initials: J
authors.lastname: Yang
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Antonenko
mesh_terms: D006096:Gramicidin / Q000737:chemistry* / Q000528:radiation effects; D015640:Ion Channel Gating / Q000528:radiation effects*; D008024:Ligands; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry*; D010777:Photochemistry / Q000379:methods; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D008024:Ligands; D008051:Lipid Bilayers; D006096:Gramicidin