Role of Gln114 in Spectral Tuning of a Long-Wavelength Sensitive Visual Pigment

Visual pigments of the long-wavelength sensitive opsin group (L group) are anion sensitive in nature. Their highly conserved amino acid residues, H197 and K200, exclusively interact with a chloride ion (Cl-) in the chromophore-binding pocket. Substitution of H197 completely abolishes Cl- binding and results in an similar to 30 nm spectral blue shift. Recent attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy studies of monkey green sensitive pigment have provided insights into the role of Cl- binding in stabilizing the antiparallel beta-sheet at extracellular loop 2 (ECL2). In addition to maintaining the dark state of L opsins, Cl- binding is also believed to play a crucial role in spectral tuning. Here, we used a combination of site-directed mutagenesis in combination with UV-visible spectroscopy to show that Q114(2.65) that is positioned far from ECL2 is also a crucial residue for the Cl- effect in L opsins. Comprehensive FTIR spectroscopic analyses on both ion-binding-induced and light-induced structural changes revealed that Q114(2.65) contributes to the stability of beta-sheet structure indirectly even though Q114(2.65) is not located in ECL2. Overall, these structure function studies are important for understanding the functional role of Cl- binding in L opsins.