Effect of Streptavidins with Varying Biotin Binding Affinities on the Properties of Biotinylated Gramicidin Channels.

摘要

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicativeof its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.