Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1Hiyoshi, Kohoku-ku, Kanagawa 223-8522, Japan.
Biochemistry. 2011 Feb 22;50(7):1174-83. doi: 10.1021/bi101689b. Epub 2011 Jan 28.
Phototropin, a blue-light receptor protein of plants, triggers phototropic responses, chloroplast relocation, and opening of stomata to maximize the efficiency of photosynthesis. Phototropin is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) that absorb blue light and a serine/theroine kinase domain responsible for light-dependent autophosphorylation leading to cellular signaling cascades. Although the light-activated LOV2 domain is primarily responsible for subsequent activation of the kinase domain, it is unclear how conformational changes in the former transmit to the latter. To understand this molecular mechanism in Arabidopsis phototropin 2, we performed small-angle X-ray scattering analysis on a fragment composed of the LOV2 and kinase domains, which contained an Asp720Asn mutation that led to an absence of ATP binding activity. The scattering data were collected up to a resolution of 25 Å. The apparent molecular weight of the fragment estimated from scattering intensities demonstrated that the fragment existed in a monomeric form in solution. The fragment exhibited photoreversible changes in the scattering profiles, and the radii of gyration under dark and blue-light irradiation conditions were 32.4 and 34.8 Å, respectively. In the dark, the molecular shape restored from the scattering profile appeared as an elongated shape of 110 Å in length and 45 Å in width. The homology modeled LOV2 and kinase domains could be fitted to the molecular shape and appeared to make slight contact. However, under blue-light irradiation, a more extended molecular shape was observed. The changes in the molecular shape and radius of gyration were interpreted as a light-dependent positional shift of the LOV2 domain of approximately 13 Å from the kinase domain. Because the region connecting the LOV2 and kinase domains was categorized as a naturally unfolded polypeptide, we propose that the light-activated LOV2 domain triggers conformational changes in the linker region to separate the LOV2 and kinase domains.
光受体蛋白（phototropin）是一种植物中的蓝光受体蛋白，它能引发向光性反应、叶绿体移位和气孔开启，以最大限度地提高光合作用的效率。光受体蛋白由两个光氧电压感应结构域（LOV1 和 LOV2）组成，它们吸收蓝光，以及一个丝氨酸/苏氨酸激酶结构域，负责光依赖性的自身磷酸化，从而引发细胞信号级联反应。虽然光激活的 LOV2 结构域主要负责激酶结构域的后续激活，但目前尚不清楚前者的构象变化如何传递到后者。为了了解拟南芥光受体蛋白 2 中的这种分子机制，我们对由 LOV2 和激酶结构域组成的片段进行了小角度 X 射线散射分析，该片段包含一个导致 ATP 结合活性缺失的 Asp720Asn 突变。散射数据的采集分辨率高达 25Å。从散射强度估算出的片段表观分子量表明，该片段在溶液中以单体形式存在。该片段表现出光致可逆的散射谱变化，在黑暗和蓝光照射条件下的回转半径分别为 32.4 和 34.8Å。在黑暗中，从散射谱中恢复的分子形状呈现出 110Å 的长度和 45Å 的宽度的拉长形状。同源建模的 LOV2 和激酶结构域可以拟合到分子形状上，并且似乎有轻微的接触。然而，在蓝光照射下，观察到更伸展的分子形状。分子形状和回转半径的变化被解释为 LOV2 结构域相对于激酶结构域的光依赖性位置移动约 13Å。由于连接 LOV2 和激酶结构域的区域被归类为天然无规卷曲多肽，因此我们提出，光激活的 LOV2 结构域引发连接区的构象变化，从而使 LOV2 和激酶结构域分离。
