Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila
Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila
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DOI：
10.1021/acs.biochem.6b00547
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发表时间：
2016-11-08
期刊：
影响因子：
2.9
通讯作者：
Larsen, Delmar S.
中科院分区：
文献类型：
--
作者：
Mix, L. Tyler;Kirpich, Julia;Larsen, Delmar S.
我们研究了双歧钩端螺旋体（Leptospira biflexa, lbiif）光活性黄蛋白（PYP）新同源物的光异构化机制，以确定该光感受器家族的主要光化学特征和功能多样性。与Halorhodospira halophila （Hhal）的原型PYP密切一致，我们观察到375 nm附近的激发态吸光度和500 nm附近的受激发射，具有三相激发态衰减。虽然libif PYP的激发态衰减是已知的PYP中最慢的，这是由于三个衰减分量的振幅的重新分布，但产生光循环进入的量子产率与Hhal PYP非常相似。Pro68在PYPs中高度保守，对Hhal PIT的高光化学量子产率很重要，但在野生型lbiif PIT中是Ile残基。I68P lbiif PYP的光产物形成水平略有增加，表明该残基调控了整个PIT家族的光化学量子产率。libif PYP还展示了一种蓝移光产物，这是以前在PYP的超快研究中未发现的，我们将其命名为pUV。我们假设pUV是扭曲质子化pCAH结构的PIT光循环中的一个弯路。用Hhal PIT进行的动力学实验证实了pUV的存在，但在室温超快实验中，这种状态的数量非常少。这些结果解决了长期以来文献中关于PYP室温光循环中存在分岔的不一致。
We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PIT, but this residue is Ile in wild-type Lbif PIT. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PIT family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PIT photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PIT confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.