Department of Chemistry, Biophysical Chemistry, Bielefeld University, Universitatsstrasse 25, 33615 Bielefeld, Germany.
Biochemistry. 2010 Feb 9;49(5):1024-32. doi: 10.1021/bi9016044.
The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved J alpha helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-J alpha and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the J alpha linker helix was observed in LOV1-LOV2-J alpha in agreement with previous studies of LOV2-J alpha. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the J alpha linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process.
蓝光光受体光受体在优化植物光合作用中起着至关重要的作用。在光受体的两个光、氧或电压敏感（LOV）结构域中，光刺激被黄素发色团吸收。该信号被假定通过保守的 Jα螺旋元件的解离和展开传递到丝氨酸/苏氨酸激酶结构域。我们通过傅里叶变换红外光谱研究了来自绿藻莱茵衣藻的全长光受体，以阐明蛋白质内的信号传递以及激酶的结构响应。通过将全长蛋白的信号与截断的 LOV1-LOV2-Jα和 LOV1-LOV2 的信号以及缺失突变体的信号进行比较，分配了光诱导的结构变化。在 LOV1-LOV2-Jα中观察到源自 Jα连接螺旋的螺旋失稳，这与先前对 LOV2-Jα的研究一致。全长光受体通过几个转角元件显示出可逆的全局构象变化。在缺失 Jα连接的缺失突变体中，这些变化被抑制，这归因于激酶结构域。转角结构的丧失被解释为 LOV2 结构域释放后激酶三级结构的光诱导打开。在激酶结构域中未观察到天冬氨酸或谷氨酸残基的伴随质子化变化。仅在存在光受体特征的激酶激活环的 54 个氨基酸延伸的情况下才观察到光诱导的螺旋失稳，该延伸被预测位于远离催化裂缝。该延伸的响应可能在光受体信号转导过程中起重要作用。
