Gramicidin-based channel systems for the detection of protein–ligand interaction

Abstract

To detect protein–ligand interaction a gramicidin-based sensor was developed. Biotin was tagged to the C-terminus of gramicidin (Gram-bio 1). The biotin-moiety, which faces the electrolyte, gave little effect on single-channel conductance. Streptavidin added to the electrolyte was detected by Gram-bio 1 through the monitoring channel current using the planar bilayer system. The suppression of macroscopic currents and the acceleration of their decaying time course were observed in a concentration dependent manner. In the single-channel level, however, no significant effect on the single-channel conductance and the open dwell time was observed upon addition of streptavidin. Therefore, streptavidin neither blocked the open channel nor changed the stability of the conducting dimer. Insertion of a linker between gramicidin and biotin did not change the streptavidin-sensitivity of the current reduction. We conclude that the binding of streptavidin to the Gram-bio 1 shifted the distribution of the complex from the membrane to the electrolyte and, thus, reduced the formation of conducting dimer of Gram-bio 1 in the membrane. Interaction of biotin with an anti-biotin antibody was also observed using this system, indicating that this system is applicable for the detection of protein–ligand interaction having a binding constant of ∼10 8–9 M −1 or more. Both the adamantane-tagged gramicidin for detection of β-cyclodextrin and the Strep Tag-II-tagged gramicidin for detection of streptavidin (binding constant: ∼10 5 M −1 or less) failed to respond. Thus, high-affinity ligands upon tagging to gramicidin render the gramicidin-based sensor able to execute as a real-time monitoring system for protein–ligand interaction.