title: Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.
abstract: Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.
authors: From the Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215 and.
authors: John I
authors: JI
authors: Ogren
authors: From the Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215 and.
authors: Adrian
authors: A
authors: Yi
authors: From the Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215 and.
authors: Sergey
authors: S
authors: Mamaev
authors: the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center, Houston, Texas 77030.
authors: Hai
authors: H
authors: Li
authors: the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center, Houston, Texas 77030.
authors: John L
authors: JL
authors: Spudich
authors: From the Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215 and kjr@bu.edu.
authors: Kenneth J
authors: KJ
authors: Rothschild
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D010940:Plant Proteins; D011522:Protons; D012243:Rhodopsin
keywords: Fourier Transform IR (FTIR); Ion Channel; Membrane Protein; Optogenetics; Rhodopsin