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The roles of transmembrane domain helix-III during rhodopsin photoactivation.

Ou WB, Yi T, Kim JM, Khorana HG - PLoS ONE (2011)

Bottom Line: Accessibility 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America. ouwb75@gmail.com

ABSTRACT

Background: Rhodopsin, 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 findings: We 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.

Significance: Accessibility 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.

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A secondary structure model of rhodopsin.Naturally occurring cysteines (dotted circles) and amino acid residues (P107-R135) mutated in this study are highlighted.
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pone-0017398-g001: A secondary structure model of rhodopsin.Naturally occurring cysteines (dotted circles) and amino acid residues (P107-R135) mutated in this study are highlighted.

Mentions: To determine the conformational changes of rhodopsin on light activation, a basal rhodopsin mutant was constructed replacing the six naturally-occurring free cysteine residues with the neutral amino acid alanine, to avoid ambiguity caused by signals derived from natural cysteines in rhodopsin. As shown in Figure 1, twenty-eight single-cysteine substituted mutants of helix-III (P107-R135) were generated based on the basal rhodopsin mutant (C140A/C167A/C185A/C222A/C264A/C316A). Single-cysteine substituted mutants on the background of the basal mutant were analyzed by rhodopsin chromophore formation, bleaching behavior, and Meta II decay. In addition, the accessibility of cysteine in each of these mutants was monitored by reaction of sulfhydryl group with 4-PDS in the dark and after illumination for 30 seconds. The results suggest that there is an aqueous/hydrophobic boundary in helix-III near G109 and I133. The chromophore-facing residues of Helix-III become water-accessible after photoactivation.


The roles of transmembrane domain helix-III during rhodopsin photoactivation.

Ou WB, Yi T, Kim JM, Khorana HG - PLoS ONE (2011)

A secondary structure model of rhodopsin.Naturally occurring cysteines (dotted circles) and amino acid residues (P107-R135) mutated in this study are highlighted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045455&req=5

pone-0017398-g001: A secondary structure model of rhodopsin.Naturally occurring cysteines (dotted circles) and amino acid residues (P107-R135) mutated in this study are highlighted.
Mentions: To determine the conformational changes of rhodopsin on light activation, a basal rhodopsin mutant was constructed replacing the six naturally-occurring free cysteine residues with the neutral amino acid alanine, to avoid ambiguity caused by signals derived from natural cysteines in rhodopsin. As shown in Figure 1, twenty-eight single-cysteine substituted mutants of helix-III (P107-R135) were generated based on the basal rhodopsin mutant (C140A/C167A/C185A/C222A/C264A/C316A). Single-cysteine substituted mutants on the background of the basal mutant were analyzed by rhodopsin chromophore formation, bleaching behavior, and Meta II decay. In addition, the accessibility of cysteine in each of these mutants was monitored by reaction of sulfhydryl group with 4-PDS in the dark and after illumination for 30 seconds. The results suggest that there is an aqueous/hydrophobic boundary in helix-III near G109 and I133. The chromophore-facing residues of Helix-III become water-accessible after photoactivation.

Bottom Line: Accessibility 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America. ouwb75@gmail.com

ABSTRACT

Background: Rhodopsin, 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 findings: We 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.

Significance: Accessibility 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.

Show MeSH