Gramicidin-based channel systems for the detection of protein-ligand interaction.
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 approximately 10(8-9) M(-1) or more. Both the adamantane-tagged gramicidin for detection of beta-cyclodextrin and the Strep Tag-II-tagged gramicidin for detection of streptavidin (binding constant: approximately 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.
Futaki
Shiroh
S
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. futaki@scl.kyoto-u.ac.jp
Zhang
Youjun
Y
Kiwada
Tatsuto
T
Nakase
Ikuhiko
I
Yagami
Takeshi
T
Oiki
Shigetoshi
S
Sugiura
Yukio
Y
D000218:Adamantane / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002273:Carcinogens / Q000378:metabolism*; D002462:Cell Membrane; D003505:Cyclodextrins / Q000378:metabolism*; D019281:Dimerization; D006096:Gramicidin / Q000737:chemistry*; D015640:Ion Channel Gating; D007473:Ion Channels; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D047392:beta-Cyclodextrins
D002273:Carcinogens; D003505:Cyclodextrins; D007473:Ion Channels; D008024:Ligands; D008051:Lipid Bilayers; D047392:beta-Cyclodextrins; D006096:Gramicidin; D001710:Biotin; D019809:Streptavidin; C031215:betadex; D000218:Adamantane