Chromophore incorporation, Pr, to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins
Chromophore incorporation, Pr, to Pfr kinetics, and Pfr thermal reversion of recombinant N-terminal fragments of phytochrome A and B chromoproteins
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DOI：
10.1021/bi980575x
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Schaffner, K
中科院分区：
文献类型：
--
作者：
Remberg, A;Ruddat, A;Schaffner, K
65 kDa（氨基酸 1-595）的燕麦光敏色素 A（phyA）和 66 kDa（1-596）的马铃薯 phyB（1-596）的 N 端脱辅基蛋白片段在大肠杆菌、酿酒酵母和巴斯德毕赤酵母中异源表达，并与光敏色素（P Phi B；天然发色团）和藻蓝蛋白 (PCB)，来自酵母的 phyA65 脱辅基蛋白在最初急剧上升后显示出单指数组装动力学，而来自大肠杆菌的相应脱辅基蛋白仅显示出缓慢的单指数组装。 phyB66 脱辅基蛋白结合任一发色团的速度比 phyA65 慢，具有双指数动力学。对于所有脱辅基蛋白，P Phi B 的掺入速度比 PCB 更快。N 端半部的 P-fr 形式的热稳定性与已知的全长重组光敏色素相似：燕麦 phyA65 P-fr 高度稳定，而马铃薯 phyB66P(fr) 快速转化为 P-r。因此，C 末端结构域和同源二聚体的形成均不调节该特性。相反，它是光敏色素的一个特征，表明其起源于单子叶植物或双子叶植物。 N 端 phyA65 和 phyB66 的 P-r 到 P-fr 动力学不同。 phyA65-PCB 的主要光产物 I-700 单指数衰减，P Phi B 类似物双指数衰减，而 phyB66 I-700 单指数衰减，与所包含的发色团无关。 N 端半部从 P-r 形成 P-fr 的速度比全长光敏色素更快，表明 C 端结构域参与了相对缓慢的蛋白质构象变化。
N-Terminal apoprotein fragments of oat phytochrome A (phyA) of 65 kDa (amino acids 1-595) and potato phyB of 66 kDa (1-596) were heterologously expressed in Escherichia coli and in the yeasts Saccharomyces cerevisiae and Pichia pastoris, and assembled with phytochromobilin (P Phi B; native chromophore) and phycocyanobilin (PCB), The phyA65 apoprotein from yeast showed a monoexponential assembly kinetics after an initial steep rise, whereas the corresponding apoprotein from E. coli showed only a slow monoexponential assembly. The phyB66 apoprotein incorporated either chromophore more slowly than the phyA65s, with biexponential kinetics. With all apoproteins, P Phi B was incorporated faster than PCB, The thermal stabilities of the P-fr forms of the N-terminal halves are similar to those known for the full-length recombinant phytochromes: oat phyA65 P-fr is highly stable, whereas potato phyB66P(fr) is rapidly converted into P-r. Thus, neither the C-terminal domain nor homodimer formation regulates this property. Rather, it is a characteristic of the phytochrome indicating its origin from mono- or dicots. The P-r to P-fr kinetics of the N-terminal phyA65 and phyB66 are different. The primary photoproduct I-700 of phyA65-PCB decayed monoexponentially and the P Phi B analogue biexponentially, whereas the phyB66 I-700 decayed monoexponentially irrespective of the chromophore incorporated. The formation of P-fr from P-r is faster with the N-terminal halves than with the full-length phytochromes, indicating an involvement of the C-terminal domain in the relatively slow protein conformational changes.