Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues

  • Shen, Yi-Chung
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Sasaki, Toshikazu
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Matsuyama, Take
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Yamashita, Takahiro
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Shichida, Yoshinori
    Department of Biophysics, Graduate School of Science, Kyoto University・Research Organization of Science and Technology, Ritsumeikan University
  • Okitsu, Takashi
    Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University
  • Yamano, Yumiko
    Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University
  • Wada, Akimori
    Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University
  • Ishizuka, Toru
    Department of Developmental Biology and Neuroscience, Graduate School of Life Science, Tohoku University
  • Yawo, Hiromu
    Department of Developmental Biology and Neuroscience, Graduate School of Life Science, Tohoku University
  • Imamoto, Yasushi
    Department of Biophysics, Graduate School of Science, Kyoto University

Description

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all-trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3, 4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6–C7, C10–C11, and C14–C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Journal

  • Biochemistry

    Biochemistry 57 (38), 5544-5556, 2018-09-25

    American Chemical Society (ACS)

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