Monovalent and multivalent binding of streptavidin to biotinylated gramicidin affects the kinetic properties of the ion channel.
Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (tau) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in tau is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of tau. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in tau was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.
Antonenko
Y N
YN
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. antonen@genebee.msu.ru
Rokitskaya
T I
TI
Kotova
E A
EA
Agapov
I I
II
Tonevitsky
A G
AG
D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001360:Avidin / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D006096:Gramicidin / Q000737:chemistry* / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D007473:Ion Channels; D008051:Lipid Bilayers; D008055:Lipids; D001360:Avidin; D006096:Gramicidin; D001710:Biotin; D019809:Streptavidin