pH-dependent bending in and out of purple membranes comprising BR-D85T.
The light-driven proton pump bacteriorhodopsin (BR) embedded in a purple membrane (PM) from Halobacterium salinarum undergoes a series of conformational changes while transporting a proton from the cytoplasmic to the extracellular side over the course of the so-called photocycle. Wild-type BR variant D85T, where aspartic acid 85 is replaced by threonine, allows for the study of structural intermediates of this photocycle that are formed in a light-dependent manner in the wild-type and in thermal equilibrium by tuning the pH of the D85T purple membrane suspension. Especially the last and least studied O-intermediate of the photocycle of bacteriorhodopsin has caught recent attention. First AFM images of D85T under acidic conditions resembling wild-type BR under physiological conditions in the O-photocycle-intermediate are presented. Bacteriorhodopsins embedded in the strongly bent purple membranes were analyzed by single molecule force spectroscopy (SMFS) providing the first single molecule force spectra of BR in the O-intermediate. SMFS was further employed to determine the absolute sign of membrane curvature. Complementary electrostatic force microscopy (EFM) was performed to support PM side discrimination and determination of the bending direction. Bending of PM-D85T was analyzed in more detail providing further insight into the structure-function relationship of the bacteriorhodopsin proton pump as well as PM behaviour at the solid-liquid junction. Findings reported here are of general interest to the field of chemomechanical transducers.
Philipps-University of Marburg, Department of Chemistry, Hans-Meerwein-Str. Bldg. H, D-35032 Marburg, Germany.
R-P
RP
Baumann
J
J
Eussner
N
N
Hampp
D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019617:Halobacterium salinarum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D018717:Purple Membrane / Q000737:chemistry / Q000378:metabolism*
D001436:Bacteriorhodopsins