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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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Vacuum electrostatics analysis of rhodopsin surface charge shows that hydrophobic/hydrophilic phase boundaries exist at position G109 of the intercellular domain.A) Left: cartoon and stick model of rhodopsin (inactive) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (inactive) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in the cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. B) Left: the cartoon and stick model of rhodopsin (partially active) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (partially active) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. Bar (bottom) shows the negative and positive charge.
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pone-0017398-g007: Vacuum electrostatics analysis of rhodopsin surface charge shows that hydrophobic/hydrophilic phase boundaries exist at position G109 of the intercellular domain.A) Left: cartoon and stick model of rhodopsin (inactive) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (inactive) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in the cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. B) Left: the cartoon and stick model of rhodopsin (partially active) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (partially active) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. Bar (bottom) shows the negative and positive charge.

Mentions: The big group including L112C-A132C/Basal mutant showed no reaction at all using prolonged times, but G109C/Basal and I133C showed completely reaction in the dark, which confirmed that hydrophobic/hydrophilic phase boundaries exist at positions of G109 and I133 of Helix-III. To determine whether the cysteine accessibility data agree with the accepted rhodopsin 3-dimensional structural model, two mutants GI09C/Basal mutant and I133C/Basal mutant were analyzed by vacuum electrostatics of rhodopsin surface charge changes in the inactive and partially active states (Figure 7 and Figure 8). The analysis data clearly demonstrated that hydrophobic/hydrophilic phase boundaries exist at positions of G109 and I133, which is consistent with the above-mentioned results. Furthermore, compared to the position changes at G109 and I133 between inactive and partially active structure, the helix-III showed little to no movement (Figure 7 and Figure 8).


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

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

Vacuum electrostatics analysis of rhodopsin surface charge shows that hydrophobic/hydrophilic phase boundaries exist at position G109 of the intercellular domain.A) Left: cartoon and stick model of rhodopsin (inactive) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (inactive) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in the cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. B) Left: the cartoon and stick model of rhodopsin (partially active) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (partially active) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. Bar (bottom) shows the negative and positive charge.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045455&req=5

pone-0017398-g007: Vacuum electrostatics analysis of rhodopsin surface charge shows that hydrophobic/hydrophilic phase boundaries exist at position G109 of the intercellular domain.A) Left: cartoon and stick model of rhodopsin (inactive) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (inactive) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in the cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. B) Left: the cartoon and stick model of rhodopsin (partially active) with the basal mutants in gray and G109C in purple. Black lines show the hydrophobic/hydrophilic phase boundaries. Number 1–7 show the transmembrane helices. Right: vacuum electrostatics model of rhodopsin (partially active) shows the highly charged hydrophilic phase (negative in red and positive in blue) both in cytoplasmic and intracellular areas and the low/non charged hydrophobic phase (between the yellow lines) in the membrane bilayer area. Purple-blue dots show the location of G109. Bar (bottom) shows the negative and positive charge.
Mentions: The big group including L112C-A132C/Basal mutant showed no reaction at all using prolonged times, but G109C/Basal and I133C showed completely reaction in the dark, which confirmed that hydrophobic/hydrophilic phase boundaries exist at positions of G109 and I133 of Helix-III. To determine whether the cysteine accessibility data agree with the accepted rhodopsin 3-dimensional structural model, two mutants GI09C/Basal mutant and I133C/Basal mutant were analyzed by vacuum electrostatics of rhodopsin surface charge changes in the inactive and partially active states (Figure 7 and Figure 8). The analysis data clearly demonstrated that hydrophobic/hydrophilic phase boundaries exist at positions of G109 and I133, which is consistent with the above-mentioned results. Furthermore, compared to the position changes at G109 and I133 between inactive and partially active structure, the helix-III showed little to no movement (Figure 7 and Figure 8).

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