Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan.
Division of Biophysics, Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan.
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):8562-8567. doi: 10.1073/pnas.1704391114. Epub 2017 Jul 24.
The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.
光激活腺苷酸环化酶（PAC）来源于光合蓝藻（OaPAC），通过 BLUF 结构域内的黄素发色团来检测光。BLUF 结构域存在于许多不同的光激活蛋白中，但相对取向不同。OaPAC 的两个 BLUF 结构域彼此紧密接触，在它们的界面形成一个螺旋卷曲。结晶不会阻碍酶的活性转换，但将晶体快速冷却至低温会阻止光激活/失活时发生的特征光谱变化。通过在光暴露后立即对晶体进行低温冷却，实现了完全激活状态下 OaPAC 的高分辨率晶体学分析。比较同晶光态和暗态结构表明，活性位点几乎没有变化，但酶活性可能会增加高达 50 倍，具体取决于条件。OaPAC 模型将有助于开发通过光提高活细胞环腺苷酸（cAMP）水平的简单、直接方法，以及其他用于光遗传学的工具。
