pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.
pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.
复制标题
pH 诱导的菠菜铁氧还蛋白构象变化：稳态和时间分辨荧光研究。
DOI：
10.1016/0003-9861(92)90094-d
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发表时间：
1992
影响因子：
3.9
通讯作者：
Hind,G
中科院分区：
文献类型：
--
作者：
Kieleczawa,J;France,LL;Sutherland,JC;Hind,G
使用稳态和时间分辨荧光技术来监测菠菜铁氧还蛋白中 pH 诱导的构象变化。色氨酸 73 (Trp-73) 发射的最大波长从 pH 6.0 以下的 325 nm 增加到 pH 7.0 以上的 342 nm，表明在吲哚环环境中，pH 7.0 时疏水性显着降低。将溶液 pH 值从 6.0 提高到 7.6 也降低了去污剂 Brij-96 的结合，表明铁氧还蛋白分子作为一个整体在较高 pH 下变得更加亲水。非离子（丙烯酰胺）和离子（I− 和 Cs+）猝灭剂用于探测色氨酸环境。 Trp-73 部分与 I− 屏蔽，可能是通过带负电荷的残基屏蔽的，正如根据植物型铁氧还蛋白的氨基酸序列和三维结构预测的那样。离子强度和 pH 对色氨酸荧光寿命的影响遵循单色氨酸蛋白质常见的模式：发射衰减可以符合双指数模型，其中激发态的寿命随着 pH 的增加而增加。参考铁氧还蛋白与铁氧还蛋白：NADP+氧化还原酶的生理关联以及光照下叶绿体基质pH值的升高，讨论了pH 6.0至7.6范围内pH诱导的构象变化的指示。
Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I−and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I−, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+oxidoreductase and the rise in chloroplast stromal pH in the light.