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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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Analysis of UV-vis spectra of cysteine mutants E113C/WT, E113/Basal mutant, and G114C/WT.Mutants were purified from COS-1 cells after regeneration with 25 µM 11-cis-retinal. UV-vis spectra were recorded in the dark, after acidification, and after illuminating the samples for 30 sec at 20°C.
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pone-0017398-g002: Analysis of UV-vis spectra of cysteine mutants E113C/WT, E113/Basal mutant, and G114C/WT.Mutants were purified from COS-1 cells after regeneration with 25 µM 11-cis-retinal. UV-vis spectra were recorded in the dark, after acidification, and after illuminating the samples for 30 sec at 20°C.

Mentions: As shown in Table 1, most of the purified-mutants/Basal mutant except for P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant formed the typical rhodopsin-like chromophore with A280/A500 ratios between 1.7 and 2.5, with an absorption λmax in the visible range varying from 480 nm to 496 nm. The A280/A500 ratio of mutants G109C/Basal mutant and R135C/Basal mutant was slightly higher, about 2.5. For mutant E113C/Basal mutant, the λmax of its chromophore was 380 nm. Upon illumination, all mutants except for P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant formed the characteristic Meta II intermediates. However, the bleaching behavior was incomplete after illuminating the samples for 30 sec, in contrast to the basal mutant. After acidification, most of the bleached samples formed the 440 nm absorbing protonated retinyl Schiff base, but the absorption λmax of E113 C/Basal mutant and G121C/Basal mutant was changed to 390 nm (Figure 2 and 3). The purified rhodopsins were homogeneous as judged by 10% SDS-PAGE (Figure S2).


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

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

Analysis of UV-vis spectra of cysteine mutants E113C/WT, E113/Basal mutant, and G114C/WT.Mutants were purified from COS-1 cells after regeneration with 25 µM 11-cis-retinal. UV-vis spectra were recorded in the dark, after acidification, and after illuminating the samples for 30 sec at 20°C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045455&req=5

pone-0017398-g002: Analysis of UV-vis spectra of cysteine mutants E113C/WT, E113/Basal mutant, and G114C/WT.Mutants were purified from COS-1 cells after regeneration with 25 µM 11-cis-retinal. UV-vis spectra were recorded in the dark, after acidification, and after illuminating the samples for 30 sec at 20°C.
Mentions: As shown in Table 1, most of the purified-mutants/Basal mutant except for P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant formed the typical rhodopsin-like chromophore with A280/A500 ratios between 1.7 and 2.5, with an absorption λmax in the visible range varying from 480 nm to 496 nm. The A280/A500 ratio of mutants G109C/Basal mutant and R135C/Basal mutant was slightly higher, about 2.5. For mutant E113C/Basal mutant, the λmax of its chromophore was 380 nm. Upon illumination, all mutants except for P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant formed the characteristic Meta II intermediates. However, the bleaching behavior was incomplete after illuminating the samples for 30 sec, in contrast to the basal mutant. After acidification, most of the bleached samples formed the 440 nm absorbing protonated retinyl Schiff base, but the absorption λmax of E113 C/Basal mutant and G121C/Basal mutant was changed to 390 nm (Figure 2 and 3). The purified rhodopsins were homogeneous as judged by 10% SDS-PAGE (Figure S2).

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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