Photokinetic, Biochemical and Structural Features of Chimeric Photoactive Yellow Protein Constructs
Photokinetic, Biochemical and Structural Features of Chimeric Photoactive Yellow Protein Constructs
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DOI：
10.1111/j.1751-1097.2012.01235.x
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发表时间：
2013-03-01
影响因子：
3.3
通讯作者：
Cusanovich, Michael A.
中科院分区：
文献类型：
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作者：
Kyndt, John A.;Meyer, Terry E.;Cusanovich, Michael A.
在已经表征的10种光活性黄色蛋白（PYP）中，与原型Halorhodospira halophila PYP相比，来自Rhodobacter物种的两种是仅有的在静息状态下具有额外中间光谱形式（max= 375 nm）的蛋白。我们构建了三个嵌合PYP蛋白，从N-末端的前21个残基（Hyb 1 PYP），从45环（Hyb 2 PYP）和两者（Hyb 3 PYP）在HhaI PYP与Rb的取代。荚膜菌PYP N-末端嵌合体在光谱和动力学上都表现得像HhaI PYP，表明Rcaps N-末端折叠在HhaI PYP的核心上。一小部分显示二聚化和较慢的恢复，可能是由于在N-末端的相互作用。环嵌合体具有少量的中间光谱形式和比Hhal PYP慢20000倍的光循环。第三个嵌合体，两个区域交换，类似于Rcaps PYP，具有大量的中间光谱形式（max= 380 nm），但具有更慢的动力学。在双嵌合体中，这些效应并不是严格的累加效应，这表明干扰一个位点的东西也会影响另一个位点。这些嵌合体表明，中间光谱形式具有其起源于整体蛋白质稳定性和溶剂暴露。
Of the 10 photoactive yellow protein (PYPs) that have been characterized, the two from Rhodobacter species are the only ones that have an additional intermediate spectral form in the resting state (max=375nm), compared to the prototypical Halorhodospira halophila PYP. We have constructed three chimeric PYP proteins by replacing the first 21 residues from the N-terminus (Hyb1PYP), 10 from the 45 loop (Hyb2PYP) and both (Hyb3PYP) in Hhal PYP with those from Rb. capsulatus PYP. The N-terminal chimera behaves both spectrally and kinetically like Hhal PYP, indicating that the Rcaps N-terminus folds against the core of Hhal PYP. A small fraction shows dimerization and slower recovery, possibly due to interaction at the N-termini. The loop chimera has a small amount of the intermediate spectral form and a photocycle that is 20000 times slower than Hhal PYP. The third chimera, with both regions exchanged, resembles Rcaps PYP with a significant amount of intermediate spectral form (max=380nm), but has even slower kinetics. The effects are not strictly additive in the double chimera, suggesting that what perturbs one site, affects the other as well. These chimeras suggest that the intermediate spectral form has its origins in overall protein stability and solvent exposure.