Rhodobacter capsulatus photoactive yellow protein:: Genetic context, spectral and kinetics characterization, and mutagenesis
Rhodobacter capsulatus photoactive yellow protein:: Genetic context, spectral and kinetics characterization, and mutagenesis
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DOI：
10.1021/bi035789f
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发表时间：
2004-02-24
期刊：
影响因子：
2.9
通讯作者：
Van Beeumen, JJ
中科院分区：
文献类型：
--
作者：
Kyndt, JA;Hurley, JK;Van Beeumen, JJ
光活性黄蛋白（PYP）的基因以前克隆从红细菌capsulatus（Rc），我们现在已经发现它与最近完成的基因组序列中的气泡形成的基因。然而，PYP尚未被表征为蛋白质。我们现在已经在大肠杆菌中产生了重组RcPYP作为谷胱甘肽-S-转移酶（GST）融合蛋白，沿着生物合成酶，导致在GST标签裂解后形成全-RcPYP。的吸收光谱（在435和375 nm处的特征峰）和光循环动力学，在445 rim处的激光闪光引发的，一般类似于球形红细菌（RsPYP），但显着不同的原型PYP从嗜盐红螺菌（HhPYP），其中有一个单一的峰值在446 nm处，并具有较慢的恢复。当用近紫外激光激发时，RcPYP也是光活性的，但终点则高于预闪光基线。这表明PYP发色团中的一些在静息状态下以顺式质子化构象存在。RcPYP中过量的435 nm形式，由重复的365 nm激光闪光形成，返回到闪光前基线，估计半衰期为2小时，这明显慢于RsPYP中相同反应的半衰期。Met 100已被报道促进HhPYP中的顺反异构化，然而Rc和RsPYP两者在位置99和100处具有Lys和Gly取代（自始至终使用HhPYP编号）并且具有比HhPYP快100倍的恢复动力学。然而，RcPYP的G100 M和K99 Q突变对动力学几乎没有影响。显然，RcPYP M100是在一个不同的构象，如最近发现的PYP结构域的百蕊杜鹃Ppr。累积结果表明，这两种红细菌PYP明显不同于已表征的其他PYP物种。这些特性还表明了不同的功能作用，我们假设它参与了气泡基因的调节，已知这些基因在其他物种中受到光调节。
A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence. However, the PYP had not been characterized as a protein. We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag. The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 rim, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery. RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline. This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state. The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP. Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP. However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics. Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr. The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized. These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.