Roles of amino acid residues near the chromophore of photoactive yellow protein
Roles of amino acid residues near the chromophore of photoactive yellow protein
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DOI：
10.1021/bi002291u
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发表时间：
2001-04-17
期刊：
影响因子：
2.9
通讯作者：
Tokunaga, F
中科院分区：
文献类型：
--
作者：
Imamoto, Y;Koshimizu, H;Tokunaga, F
为了研究发色团周围氨基酸残基在光活性黄蛋白（PYP）中的作用，制备了新的突变体Y424、E46A和T50A。将其与野生型、Y42F、E46Q、T50V、R52Q和E46Q/T50V的光谱特性进行比较，Y42A、E46A和T50A分别在438、469和454 nm处观察到最大吸收。发色团的pH滴定结果表明，PYP突变体的发色团与野生型一样，在酸性条件下被质子化和漂白。突变体吸收最大值的红移倾向于pK(a)的增加。Glu46突变引起了吸收最大值和pK(a)的显著变化。消光系数随吸收最大值成比例增加，而振荡器强度不变。保守Tyr42的PYP突变体处于两种状态（黄色和无色形式）之间的ph依赖平衡状态。然而，Y42A和Y42F在中性pH附近处于与pH无关的中间态平衡，其中Y42F以黄色形式为主，而Y42A以黄色形式为主。这些发现表明，Tyr42作为蛋白的铰链，Tyr42的体积和羟基控制着蛋白的构象。在所有突变体中，在450nm处的吸光度在闪光灯照射下下降，然后在毫秒时间尺度上恢复。然而，340-370 nm处的吸光度增加，反之亦然，表明长寿命的近紫外中间体是由突变体形成的，就像野生型的情况一样。突变导致的寿命变化表明质子通过氢键网络运动的调控。
To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y424, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified, The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340-370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.