Kottke Tilman, Batschauer Alfred, Ahmad Margaret, Heberle Joachim
IBI-2, Structural Biology, Research Center Jülich, 52425 Jülich, Germany.
Biochemistry. 2006 Feb 28;45(8):2472-9. doi: 10.1021/bi051964b.
Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm(-)(1) in D(2)O. The main positive band at 1524 cm(-)(1) in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution.
隐花色素是蓝光光感受器，可调节动植物中的多种反应，包括果蝇的昼夜节律调节和拟南芥的光形态建成。它们由一个约500个氨基酸的光解酶同源区域（PHR）和一个长度可变的C端延伸组成。在PHR结构域中，黄素腺嘌呤二核苷酸（FAD）非共价结合。动物和植物隐花色素中是否存在第二个发色团，如亚甲基四氢叶酸，仍存在争议。关于拟南芥隐花色素1（CRY1）在体内和体外的功能及蛋白质相互作用，已经进行了深入研究。然而，在分子水平上，从光吸收到信号转导的途径仍知之甚少。我们通过傅里叶变换红外（FTIR）和紫外/可见差分光谱对全长CRY1蛋白进行了研究。从发色团FAD的完全氧化状态开始，在没有任何外源电子供体的情况下光照会形成一个中性黄素蛋白自由基。给出了发色团谱带的初步归属。FTIR差分光谱显示，响应发色团的光还原，脱辅基蛋白的二级结构仅发生适度变化。从D2O中1734 cm-1处的负谱带推断，天冬氨酸或谷氨酸（可能是D396）的去质子化伴随着自由基的形成。与其他黄素蛋白相比，FTIR光谱中1524 cm-1处的主要正谱带向低频方向有强烈位移。再加上可见范围内吸收峰异常蓝移至595 nm，这清楚地将CRY1的自由基形式与结构高度同源的DNA光解酶的自由基形式区分开来。因此，在光反应性和机制方面将隐花色素与光解酶进行直接比较时必须谨慎。
