Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy
Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy
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DOI：
10.1021/ja038224f
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发表时间：
2004-03-24
影响因子：
15
通讯作者：
Gardner, KH
中科院分区：
文献类型：
--
作者：
Harper, SM;Neil, LC;Gardner, KH
向光蛋白是来自植物的光激活激酶，其利用光-氧-电压（LOV）结构域作为蓝光光传感器。这些结构域的发光导致蛋白质和内部结合的黄素发色团之间形成共价键，使周围蛋白质不稳定并从其表面置换α-螺旋。在这里，我们使用光谱工具的组合，以监测自发发生在黑暗中的蛋白质返回到非共价基态的动力学过程。使用时间分辨二维（2D）NMR方法，我们测量了整个蛋白质中超过100个独立位点的这一过程的速率，确定了暗态再生在1.6倍的观测速率范围内协同发生。这些数据同意与其他光谱测量的蛋白质/FMN键裂解和全球构象变化的动力学，与这些过程经历一个共同的限速步骤一致。这些速率的温度依赖性的阿克里尼乌斯分析表明，在这个再生过程中访问的过渡态具有更高的能量比变性形式的蛋白质结构域，尽管事实上，没有全球展开的域在这个过程中。
Phototropins are light-activated kinases from plants that utilize light−oxygen−voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an α-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.