title: Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1.
abstract: Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2(380) intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H2 (18)O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm(-1)) and with the other O-H group medium (3440 cm(-1)) to moderately strongly (3300 cm(-1)) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.affiliation: Genetic Biophysics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Muders
authors.affiliation: Genetic Biophysics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Schlesinger
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
mesh_terms: D002696:Chlamydomonas / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D006860:Hydrogen Bonding; D007473:Ion Channels / Q000737:chemistry*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry*; D025604:Rhodopsins, Microbial / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water / Q000737:chemistry*
chemical_list: D007473:Ion Channels; D010940:Plant Proteins; D025604:Rhodopsins, Microbial; D014867:Water; D003545:Cysteine