Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation
Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation
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DOI：
10.1039/c2pp25222h
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发表时间：
2013-01-01
影响因子：
3.1
通讯作者：
Hellingwerf, Klaas J.
中科院分区：
文献类型：
--
作者：
Hospes, Marijke;Hendriks, Johnny;Hellingwerf, Klaas J.
光敏黄色蛋白（PYP）的光激活之后会发生一系列蛋白质结构的动态转变。色氨酸荧光非常适合作为研究这些选定方面的工具。使用定点诱变，通过用色氨酸替换酪氨酸、苯丙氨酸或组氨酸残基，制备了 PYP 的八个“单色氨酸”突变体，同时消除了内源性 W119。令人惊讶的是，这八个突变体中只有三个发出可测量的色氨酸荧光：F6W/W119F、F96W/W119F 和 H108W/W119F。值得注意的是，在 pB 状态形成时，所有三种颜色都显示出色氨酸荧光的改变。由于F96非常靠近发色团，因此F96W/W119F突变蛋白特别适合进一步研究PYP发色团结合口袋中极性的动态变化。此外，WT PYP 可以通过高度保守的 W119 和随后的福斯特共振能量转移被 UV 光子光激活。将独特的色氨酸残基放置在蛋白质的其他位置表明位置 119 有利于 PYP 的紫外线激活。
Light-activation of photoactive yellow protein (PYP) is followed by a series of dynamical transitions in the structure of the protein. Tryptophan fluorescence is well-suited as a tool to study selected aspects of these. Using site-directed mutagenesis eight 'single-tryptophan' mutants of PYP were made by replacement of either a tyrosine, phenylalanine or histidine residue by tryptophan, while simultaneously eliminating the endogenous W119. Surprisingly, only three of these eight mutants turn out to emit measurable tryptophan fluorescence: F6W/W119F, F96W/W119F and H108W/W119F. Significantly, all three show altered tryptophan fluorescence upon formation of the pB state. As F96 is located very close to the chromophore, the F96W/W119F mutant protein is particularly suitable for further studies on the dynamical changes of the polarity in the chromophore-binding pocket of PYP. Furthermore, WT PYP can be photo-activated by a UV photon via the highly conserved W119 and subsequent Forster resonance energy transfer. Placing a unique tryptophan residue elsewhere in the protein shows that position 119 is favoured for UV-activation of PYP.