Macromolecular crowding effect for photoreactions of LOV2 domains of Arabidopsis thaliana phototropin 1
Macromolecular crowding effect for photoreactions of LOV2 domains of Arabidopsis thaliana phototropin 1
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拟南芥趋光素1 LOV2结构域光反应的大分子拥挤效应
DOI：
10.1016/j.molliq.2015.08.030
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发表时间：
2016
影响因子：
6
通讯作者：
Masahide Terazima
中科院分区：
文献类型：
--
作者：
Tomoyuki Yoshitake;Tsuguyoshi Toyooka;Yusuke Nakasone;Kazunori Zikihara;Satoru Tokutomi;Masahide Terazima
Phototropins（phot 1和phot 2）是蓝光传感器，由两个N-末端LOV（光-氧-电压传感）结构域（LOV 1和LOV 2）、C-末端的丝氨酸/苏氨酸激酶结构域和连接这些结构域的接头区组成。phot 1 LOV 2结构域和phot 1 LOV 2-linker结构域的化学反应主要在缓冲溶液中进行研究。在这项研究中，拥挤效应的光反应的phot 1 LOV 2和phot 1 LOV 2-连接器结构进行了研究。通过紫外/可见吸收光谱和圆二色性光谱监测的两种样品在暗态下的构象不受拥挤剂Ficoll-70的存在的影响。相反，随着Ficoll-70浓度的增加，观察到代表phot 1 LOV 2二聚体解离反应的瞬态光栅信号增加，并且phot 1 LOV 2单体的二聚反应减少。这种效果被解释在拥挤的条件下，在黑暗中从单体到二聚体的平衡位移。时间依赖性圆二色性信号表明，在拥挤试剂存在下，phot 1 LOV 2-接头的反应保持不变，即，连接体在光激发时解折叠。然而，随着Ficoll-70浓度的增加，反应速率常数略有下降。尽管在200 g/L的Ficoll-70浓度下粘度增加约10倍，但速率常数仅降低1.5倍。这种对拥挤效应的明显不敏感性可以通过不受拥挤剂如Ficoll-70阻碍的接头的局部结构变化来解释。
Phototropins (phot1 and phot2) are blue light sensors consisting of two N-terminal LOV (light–oxygen–voltage sensing) domains (LOV1 and LOV2), a serine/threonine kinase domain at the C-terminus, and a linker region connecting these domains. The chemical reactions of the phot1LOV2 domain and phot1LOV2-linker domain have been studied mostly in buffer solutions. In this study, crowding effects on photoreactions of phot1LOV2 and phot1LOV2-linker constructs were investigated. The conformations of both samples in the dark states monitored by ultraviolet/visible absorption and circular dichroism spectra were not influenced by the presence of the crowding agent Ficoll-70. Conversely, with increasing concentrations of Ficoll-70, the transient grating signal representing the dissociation reaction of the phot1LOV2 dimer was observed to increase, and the dimerization reaction of the phot1LOV2 monomer decreased. This effect was interpreted in terms of an equilibrium shift from the monomer to the dimer in the dark under crowding conditions. The time-dependent circular dichroism signal indicated that the reaction of phot1LOV2-linker remained unchanged in the presence of the crowding reagent, i.e., the linker is unfolded upon photoexcitation. However, the reaction rate constant decreased slightly with increasing concentration of Ficoll-70. Although the viscosity increased ~ 10-fold at a Ficoll-70 concentration of 200 g/L, the rate constant decreased only by 1.5-fold. This apparent insensitivity to crowding effects is explained by local structural changes to the linker that are not obstructed by crowding agents such as Ficoll-70.