Abstract
BackgroundRhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear.Principal FindingsWe herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4′-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees.SignificanceAccessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.
Highlights
G-protein-coupled receptors (GPCRs), the largest known family of cell surface receptors, mediate a wide variety of signal transduction processes
The mechanism by which rhodopsin is activated has been extensively characterized [2,3,4,5,6,7,8,9,10,11,12,13,14]; the most relevant studies in this regard include the recently determined crystal structure of inactive [2] and partially activate rhodopsin [7,8]; site-directed spin labeling and double electron-electron resonance (DEER) studies [9]; the model of two protonation switches operating at the Schiff base (E113) and the cytosol (E134) [10,11]; and the function of structural waters [12,13,14].the role of TM helix-III in rhodopsin photoactivation remains unclear
Due to difficulties of getting the crystal structure of completely activated rhodopsin, several biochemical approaches have been employed to analyze its structural changes upon activation
Summary
G-protein-coupled receptors (GPCRs), the largest known family of cell surface receptors, mediate a wide variety of signal transduction processes. It is well known that light induces rhodopsin isomerization from 11-cis-retinal to all-transretinal. This isomerization activates the receptor by causing movements of the TM helices, which, in turn, induce conformational changes in the CP domain that result in transition to the activated state [16]. This state is competent for binding the heterotrimeric G-protein of the rod cell transducin (Gt), and for catalysis of the uptake of guanosine triphosphate by the a-subunit of Gt, thereby initiating the enzymatic cascade that leads to light detection and to vision. The basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear
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