Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant

Megan N Sandberg; Jordan A Greco; Nicole L Wagner; Tabitha L Amora; Lavoisier A Ramos; Min-Hsuan Chen; Barry E Knox; Robert R Birge

doi:10.15226/2376-4589/1/1/00103

Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant

SOJ Biochem. 2014;1(1):12. doi: 10.15226/2376-4589/1/1/00103.

Authors

Megan N Sandberg¹, Jordan A Greco¹, Nicole L Wagner¹, Tabitha L Amora¹, Lavoisier A Ramos¹, Min-Hsuan Chen², Barry E Knox², Robert R Birge¹

Affiliations

¹ Departments of Chemistry and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
² Departments of Biochemistry and Molecular Biology and Ophthalmology State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

Abstract

Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λ_max = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λ_max = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λ_max = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.

Keywords: Absorption spectroscopy; Blue-Shifted Intermediate (BSI); E181Q; Glu-181; Low-Temperature trapping; Photobleaching sequence; Photointermediates; Rhodopsin.

Abstract

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