Purchase this article with an account.
M.–H. Chen, L. Ramos, K. Babu, R.R. Birge, B.E. Knox; The Role of Electrostatics in the Photoactivation of a Violet Cone Visual Pigment . Invest. Ophthalmol. Vis. Sci. 2006;47(13):805.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To better understand the role of electrostatic interactions between the protonated retinylidene Schiff base and the conserved acidic residue that acts as the primary Schiff base counterion, we characterized the photoactivation pathway in several mutants of Xenopus violet cone opsin (VCOP).
Site–specific mutants in VCOP were prepared and expressed in COS1 cells using transient transfection. Purified protein in 0.03% dodecyl–maltoside, 67% glycerol containing varying buffers were used for cryogenic spectroscopic experiments. The homology models of various VCOP protein were created based on the crystal structure of bovine rhodopsin (1U19), and embedded into a lipid bilayer and solvent water containing ions of sodium and chloride. Molecular dynamics (NAMD) was utilized on the whole system for 2 ns to prepare the final model.
D108 serves as the primary counterion that interacts with the Schiff base in the dark (Babu et al., 2001, Biochemisty, 40: 760–6). In the dark, the D108A mutant had an unprotonated Schiff base (max 363 nm) vs. wild type (max 425 nm). After illumination (70 K), a batho intermediate (max 367 nm) vs. wild type (max 450 nm) formed. Above 210 K, two species formed, unprotonated lumi I (max 390 nm) and protonated lumiII (max 460 nm) compared to wild type (max 435 nm). There was temperature, time, and pH dependent conversion of lumiI to lumiII. LumiII further decayed >233 K into meta I (max 440 nm) vs. wild type (max 420 nm). Above 263 K metaII formed (max 365 nm) similar to wild type. The D108A/S85D mutant had a very broad absorption (max ∼400 nm), but formed a batho intermediate similar to wild type VCOP after illumination at 70 K. Only one lumi formed (max ∼440 nm) and meta I and meta II were similar to wild type. However, intermediates were less stable for the D108A/S85D mutant than wild type. Molecular modeling suggested that S85D carboxyl group is further away from the protonated Schiff base than D108 carboxyl group in wild type.
1. A protonated lumi is required to form the meta I intermediate. 2. Even when the Schiff base is initially unprotonated and the primary counterion is absent, a protonated lumi still forms although at kinetically slower rate than wild type. 3. Weakening the electrostatic interaction between the primary counterion and the Schiff base by increasing the distance between them destabilizes the photoactivation pathway.
This PDF is available to Subscribers Only