title: Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K.
abstract: A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet-visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C═O stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 → Gln and Ser97 → Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 → Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.
authors.affiliation: Molecular Biophysics Laboratory, Photonics Center, and Department of Physics, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Adrian
authors.identifier: 0000-0001-9067-724X
authors.initials: A
authors.lastname: Yi
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, McGovern Medical School , Houston, Texas 77030, United States.
authors.forename: Hai
authors.initials: H
authors.lastname: Li
authors.affiliation: Molecular Biophysics Laboratory, Photonics Center, and Department of Physics, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Natalia
authors.initials: N
authors.lastname: Mamaeva
authors.affiliation: Molecular Biophysics Laboratory, Photonics Center, and Department of Physics, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Roberto E
authors.initials: RE
authors.lastname: Fernandez De Cordoba
authors.affiliation: Department of Biophysical Organic Chemistry, Leiden Institute of Chemistry, Leiden University , 2300 AR Leiden, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Lugtenburg
authors.affiliation: Department of Biophysical Organic Chemistry, Leiden Institute of Chemistry, Leiden University , 2300 AR Leiden, The Netherlands.
authors.forename: Willem J
authors.identifier: 0000-0001-7637-4920
authors.initials: WJ
authors.lastname: DeGrip
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, McGovern Medical School , Houston, Texas 77030, United States.
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
authors.affiliation: Molecular Biophysics Laboratory, Photonics Center, and Department of Physics, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Kenneth J
authors.identifier: 0000-0002-2847-6671
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D019943:Amino Acid Substitution; D000838:Anions; D003080:Cold Temperature; D005030:Ethylenes / Q000737:chemistry; D010843:Pichia / Q000737:chemistry; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
chemical_list: D000838:Anions; D005030:Ethylenes; D012243:Rhodopsin; C036216:ethylene