Direct Observation of the pH-Dependent Equilibrium between Metarhodopsins I and II and the pH-Independent Interaction of Metarhodopsin II with Transducin C-Terminal Peptide
Direct Observation of the pH-Dependent Equilibrium between Metarhodopsins I and II and the pH-Independent Interaction of Metarhodopsin II with Transducin C-Terminal Peptide
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DOI：
10.1021/bi9018412
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发表时间：
2010-02-02
期刊：
影响因子：
2.9
通讯作者：
Shichida, Yoshinori
中科院分区：
文献类型：
--
作者：
Sato, Keita;Morizumi, Takefumi;Shichida, Yoshinori
牛视紫红质含有11-顺式-视黄醛作为光吸收发色团，其通过质子化的席夫碱键与脱辅基蛋白视蛋白的赖氨酸残基结合。光将11-顺式-视黄醛异构化为全反式形式，这最终导致形成酶活性状态，即变视紫红质II（MII）。人们普遍认为，MII形成一个pH依赖性的平衡与metarhodopsin I（MI），但这种平衡的直接证据还没有报道。在这里，我们通过直接观察MI和MII在改变MI/MII混合物的pH时的相互转化来证实这种平衡。我们还观察到构成转导素α-亚基的C-末端11个氨基酸的合成肽与MII的可逆结合，这导致平衡中MI和MII的量的变化。有趣的是，除了肽没有诱导一个简单的pK（a）的变化，而是诱导增加的MII部分在高pH值。这些结果表明，除了MII是由MI在pH值依赖性的方式形成的，也存在另一个MII，这是在平衡与MI在pH值不依赖的方式，并可以结合到肽。因此，不需要通过视蛋白的蛋白质部分的质子摄取来结合肽。
Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state, metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibrium with metarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions of MI and MII upon changing the pH of the MI/MII mixture. We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin alpha-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pK(a) shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to the MII that is formed from MI in a pH-dependent manner there also exists another MII, which is in equilibrium with MI in a pH-independent manner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.