Biophysical, Mutational, and Functional Investigation of the Chromophore-Binding Pocket of Light-Oxygen-Voltage Photoreceptors
Biophysical, Mutational, and Functional Investigation of the Chromophore-Binding Pocket of Light-Oxygen-Voltage Photoreceptors
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DOI：
10.1021/sb400205x
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发表时间：
2014-11-01
影响因子：
4.7
通讯作者：
Moeglich, Andreas
中科院分区：
文献类型：
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作者：
Diensthuber, Ralph P.;Engelhard, Christopher;Moeglich, Andreas
作为光调控的致动器，感光感受器为光遗传学和合成生物学的众多应用奠定了基础。蛋白质工程已经被应用于微调光感受器的特性和产生新的致动器。对于蓝光敏感的光-氧-电压(LOV)光感受器，黄素发色团附近的突变调制了响应动力学和有效的光响应。为了探索潜在的、无意中对受体活性的影响，我们将这些突变引入到工程LOV光感受器YF1中，并确定它们对光调节的影响。虽然几个突变严重损害了受体的动态范围(例如，I39V、R63K和N94A)，但第二组中的残基替换是良性的，对调节几乎没有影响(例如，V28T、N37C和L82I)。电子顺磁共振和吸收光谱证实了后一种突变对燕麦促光素1的YF1和LOV2结构域中生色团环境和响应动力学的相关影响。精心选择的突变为调节光感受器的光响应功能提供了一种强大的手段，以满足不同应用的需要。
As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.