Engineering an inward proton transport from a bacterial sensor rhodopsin.
ATP is synthesized by an enzyme that utilizes proton motive force, and thus, nature has created various proton pumps. The best-understood proton pump is bacteriorhodopsin (BR), an outward-directed, light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. We previously converted BR into an inward-directed chloride ion pump, but an inward proton pump has never been created. Proton pumps must have a specific mechanism to exclude transport in the reverse direction in order to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Here we report that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria that possesses little proton pump activity. When we replaced Asp217 in the cytoplasmic domain (a distance of approximately 15 A from the retinal chromophore) by Glu, ASR exhibited an inward proton transport activity driven by absorption of a single photon. FTIR spectra clearly showed an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to that in normal proton pumps.
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Akira
A
Kawanabe
Yuji
Y
Furutani
Kwang-Hwan
KH
Jung
Hideki
H
Kandori
D017033:Anabaena / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008027:Light; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D017494:Proton Pumps / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
D017494:Proton Pumps; D001436:Bacteriorhodopsins