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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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Comparison of PDS labeling rate among cysteine mutants and relationship analysis of PDS labeling rate with Meta II decay.A) Rates of cysteine reactivity with 4-PDS were evaluated in mutants (F115/, F116/, A117/, T118/, V130/, L131/, A132/, I133/, and R135/Basal mutant). The reaction was carried out with 0.5 µM of rhodopsin mutant and 25 µM 4-PDS in phosphate buffer (pH8.0) and 0.05% DM at 20°C. Time-dependent changes in absorbance at 323 nm after photoactivation were plotted. B) Rates of cysteine reactivity with 4-PDS (dark gray) and Meta II decay (light gray) in relationship to the amino acid position.
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pone-0017398-g006: Comparison of PDS labeling rate among cysteine mutants and relationship analysis of PDS labeling rate with Meta II decay.A) Rates of cysteine reactivity with 4-PDS were evaluated in mutants (F115/, F116/, A117/, T118/, V130/, L131/, A132/, I133/, and R135/Basal mutant). The reaction was carried out with 0.5 µM of rhodopsin mutant and 25 µM 4-PDS in phosphate buffer (pH8.0) and 0.05% DM at 20°C. Time-dependent changes in absorbance at 323 nm after photoactivation were plotted. B) Rates of cysteine reactivity with 4-PDS (dark gray) and Meta II decay (light gray) in relationship to the amino acid position.

Mentions: Upon photoactivation, these mutants incorporated ∼1 mole of the reagent per mole of rhodopsin over time after illuminating the samples for 30 sec. After light activation, comparison of the rate of reactivity calculated from absorbance increase at 323 nm indicated that mutants T108C/, G109C/, E113C/, and L131C/Basal mutant showed rapid reactions with 4-PDS. Reaction rate of mutants L112C/, G121C/, and A132C/Basal mutant with 4-PDS was moderate, while mutants F115C/, T118C/, L119C/, V129C/, V130C/, and R135C/Basal mutant showed a slow rate of reaction with 4-PDS. The slower reactions were observed with F116C/, A117C/, G120C/, E122C/, I123C/, S127C/, and I133C/Basal mutant. The cysteine mutant E113C/Basal mutant showed the fastest reaction rate with 4-PDS among all of the mutants at helix-III. The time courses for the reactions and exponential fits for selected mutants are shown in Figure 6A and Figure S5, and the pseudo-first-order rate constants for all of the mutants are listed in Table 2 and Figure 6B.


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

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

Comparison of PDS labeling rate among cysteine mutants and relationship analysis of PDS labeling rate with Meta II decay.A) Rates of cysteine reactivity with 4-PDS were evaluated in mutants (F115/, F116/, A117/, T118/, V130/, L131/, A132/, I133/, and R135/Basal mutant). The reaction was carried out with 0.5 µM of rhodopsin mutant and 25 µM 4-PDS in phosphate buffer (pH8.0) and 0.05% DM at 20°C. Time-dependent changes in absorbance at 323 nm after photoactivation were plotted. B) Rates of cysteine reactivity with 4-PDS (dark gray) and Meta II decay (light gray) in relationship to the amino acid position.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017398-g006: Comparison of PDS labeling rate among cysteine mutants and relationship analysis of PDS labeling rate with Meta II decay.A) Rates of cysteine reactivity with 4-PDS were evaluated in mutants (F115/, F116/, A117/, T118/, V130/, L131/, A132/, I133/, and R135/Basal mutant). The reaction was carried out with 0.5 µM of rhodopsin mutant and 25 µM 4-PDS in phosphate buffer (pH8.0) and 0.05% DM at 20°C. Time-dependent changes in absorbance at 323 nm after photoactivation were plotted. B) Rates of cysteine reactivity with 4-PDS (dark gray) and Meta II decay (light gray) in relationship to the amino acid position.
Mentions: Upon photoactivation, these mutants incorporated ∼1 mole of the reagent per mole of rhodopsin over time after illuminating the samples for 30 sec. After light activation, comparison of the rate of reactivity calculated from absorbance increase at 323 nm indicated that mutants T108C/, G109C/, E113C/, and L131C/Basal mutant showed rapid reactions with 4-PDS. Reaction rate of mutants L112C/, G121C/, and A132C/Basal mutant with 4-PDS was moderate, while mutants F115C/, T118C/, L119C/, V129C/, V130C/, and R135C/Basal mutant showed a slow rate of reaction with 4-PDS. The slower reactions were observed with F116C/, A117C/, G120C/, E122C/, I123C/, S127C/, and I133C/Basal mutant. The cysteine mutant E113C/Basal mutant showed the fastest reaction rate with 4-PDS among all of the mutants at helix-III. The time courses for the reactions and exponential fits for selected mutants are shown in Figure 6A and Figure S5, and the pseudo-first-order rate constants for all of the mutants are listed in Table 2 and Figure 6B.

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
Related in: MedlinePlus