Thermochromatium tepidum photoactive yellow protein/bacteriophytochrome/diguanylate cyclase: characterization of the PYP domain.
Thermochromatium tepidum photoactive yellow protein/bacteriophytochrome/diguanylate cyclase: characterization of the PYP domain.
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Thermochromatium tepidum 光敏黄色蛋白/细菌光敏色素/二鸟苷酸环化酶：PYP 结构域的表征。
DOI：
10.1021/bi047373n
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Cusanovich,MichaelA
中科院分区：
文献类型：
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作者：
Kyndt,JohnA;Fitch,JohnC;Meyer,TerryE;Cusanovich,MichaelA
紫色光养细菌，Thermochromatium tepidum，含有一个嵌合的光活性黄色蛋白/细菌光敏色素/二鸟苷酸环化酶（Ppd）的基因。我们生产了TC。tepidumPYP结构域（Tt PYP）在大肠杆菌中的表达，并发现它的最大波长为358 nm，这是由于原型嗜盐旋菌PYP（Hh PYP）中发现的颜色调节Glu 46被Leu 46取代。然而，358 nm的暗适应状态是在一个pH依赖性的平衡与黄色物质吸收在465 nm（pKa= 10.2）。在358 nm处照射后，光循环动力学的特征在于在pH 7.0下通过小的漂白和红移到似乎是长寿命的顺式中间体（与Hh PYP中的I2中间体相当）。恢复到暗适应状态的寿命为104 min，比Hh PYP慢约1500倍。然而，当Tt PYP被照射在pH值高于7.5时，光诱导的差光谱表明I2中间体和红移440 nm的中间体之间的pH依赖性平衡。这种平衡可能是负责的S形pH依赖的恢复的暗适应状态（pKa= 8.8）。此外，光诱导的差异光谱表明，在pH值高于9.3，有一个明显的漂白附近490 nm叠加在358和440 nm的变化，我们归因于质子化和电离的黑暗适应形式之间的平衡。Tt PYP的L46 E突变体在446 nm处具有最大波长，类似于野生型Hh PYP。用白色光照射后L46 E的恢复动力学缓慢（在pH 7下的寿命为15分钟），但与野生型Tt PYP的恢复动力学相当。我们的结论是，Tt PYP是唯一的PYPs研究迄今为止，它有一个光循环开始从一个黑暗适应状态与质子化的发色团在生理pH值。然而，它是动力学最相似的tohodocista centenariaPYP（PPR），尽管非常不同的吸收光谱，由于缺乏E46。
The purple phototrophic bacterium,Thermochromatium tepidum, contains a gene for a chimeric photoactive yellow protein/bacteriophytochrome/diguanylate cyclase (Ppd). We produced theTc. tepidumPYP domain (Tt PYP) inEscherichia coli, and found that it has a wavelength maximum at 358 nm due to a Leu46 substitution of the color-tuning Glu46 found in the prototypicHalorhodospira halophilaPYP (Hh PYP). However, the 358 nm dark-adapted state is in a pH-dependent equilibrium with a yellow species absorbing at 465 nm (pKa= 10.2). Following illumination at 358 nm, photocycle kinetics are characterized at pH 7.0 by a small bleach and red shift to what appears to be a long-lived cis intermediate (comparable to the I2intermediate in Hh PYP). The recovery to the dark-adapted state has a lifetime of ∼4 min, which is approximately 1500 times slower than that for Hh PYP. However, when the Tt PYP is illuminated at pH values above 7.5, the light-induced difference spectrum indicates a pH-dependent equilibrium between the I2intermediate and a red-shifted 440 nm intermediate. This equilibrium could be responsible for the sigmoidal pH dependence of the recovery of the dark-adapted state (pKa= 8.8). In addition, the light-induced difference spectrum shows that, at pH values above 9.3, there is an apparent bleach near 490 nm superimposed on the 358 and 440 nm changes, which we ascribe to the equilibrium between the protonated and ionized dark-adapted forms. The L46E mutant of Tt PYP has a wavelength maximum at 446 nm, resembling wild-type Hh PYP. The kinetics of recovery of L46E following illumination with white light are slow (lifetime of 15 min at pH 7), but are comparable to those of wild-type Tt PYP. We conclude that Tt PYP is unique among the PYPs studied to date in that it has a photocycle initiated from a dark-adapted state with a protonated chromophore at physiological pH. However, it is kinetically most similar toRhodocista centenariaPYP (Ppr) despite the very different absorption spectra due to the lack of E46.