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.
Kawanabe
Akira
A
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Furutani
Yuji
Y
Jung
Kwang-Hwan
KH
Kandori
Hideki
H
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