The roles of transmembrane domain helix-III during rhodopsin photoactivation.
The roles of transmembrane domain helix-III during rhodopsin photoactivation.
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DOI：
10.1371/journal.pone.0017398
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发表时间：
2011-02-25
期刊：
影响因子：
3.7
通讯作者：
Khorana HG
中科院分区：
文献类型：
--
作者：
Ou WB;Yi T;Kim JM;Khorana HG
视紫红质是 G 蛋白偶联受体 (GPCR) 的原型成员，在光激活后经历 11-顺式-视黄醛异构化为全反式-视黄醛。尽管视紫红质被激活的基本机制已被充分了解，但整个跨膜（TM）螺旋-III在视紫红质光激活过程中的详细作用尚不完全清楚。我们在此使用单半胱氨酸诱变技术来研究光激活后视紫红质TM螺旋的构象变化。具体来说，我们研究了引入视紫红质 TM 螺旋 III 的半胱氨酸残基的可及性和反应性的变化。将所有天然半胱氨酸残基（C140/C167/C185/C222/C264/C316）替换为丙氨酸后，制备了二十八个视紫红质单半胱氨酸突变体（P107C-R135C）。半胱氨酸突变体在 COS-1 细胞中表达，视紫红质在用 11-顺式视黄醛再生后进行纯化。通过在黑暗中和光照后与 4, 4'-二硫代二吡啶 (4-PDS) 反应来监测这些突变体中的半胱氨酸可及性。除T108C、G109C、E113C、I133C和R135C外，大多数突变体在黑暗中没有表现出反应。光活化后，在序列 108-135 的不同位置上观察到半胱氨酸的反应性存在很大差异。特别是，面向11-顺式视黄醛的115、119、121、129、131、132和135位的半胱氨酸与4-PDS的反应比邻近的氨基酸更快。光活化后突变体与4-PDS的不同反应速率表明helix-III中不同位置的氨基酸不同程度地暴露于水环境。可及性数据表明螺旋-III 中的水/疏水边界位于 G109 和 I133 附近。黑暗中缺乏反应性以及光活化后半胱氨酸的可及性表明在 Meta II 形成过程中 Helix-III 处某些半胱氨酸突变体的水/4-PDS 可及性增加。我们得出结论，光活化导致 Helix-III 面向发色团的残基可与水接触。
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. 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. 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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DOI：
10.1073/pnas.90.21.10206
发表时间：
1993-11-01
影响因子：
11.1
作者：
FAHMY, K;JAGER, F;SIEBERT, F
通讯作者：
SIEBERT, F
DOI：
10.1073/pnas.83.3.599
发表时间：
1986-02-01
影响因子：
11.1
作者：
FERRETTI, L;KARNIK, SS;OPRIAN, DD
通讯作者：
OPRIAN, DD
DOI：
10.1042/bj20100270
发表时间：
2010-04-28
期刊：
The Biochemical journal
影响因子：
--
作者：
Jastrzebska B;Tsybovsky Y;Palczewski K
通讯作者：
Palczewski K
DOI：
10.1073/pnas.94.26.14267
发表时间：
1997-12-23
影响因子：
11.1
作者：
Cai, KW;Langen, R;Khorana, HG
通讯作者：
Khorana, HG
DOI：
10.1073/pnas.0710393104
发表时间：
2007-12-18
影响因子：
11.1
作者：
Knierim, Bernhard;Hofmann, Klaus Peter;Hubbell, Wayne L.
通讯作者：
Hubbell, Wayne L.