Baca M, Borgstahl G E, Boissinot M, Burke P M, Williams D R, Slater K A, Getzoff E D
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Biochemistry. 1994 Dec 6;33(48):14369-77. doi: 10.1021/bi00252a001.
The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.
光活性蛋白捕获并利用光子能量的独特能力取决于发色团、其与蛋白质的连接方式以及周围的蛋白质环境。为了理解发色团与蛋白质相互作用以经历光循环的分子机制，我们正在研究光活性黄色蛋白（PYP），它是一种来自嗜盐外硫红螺菌的14 kDa水溶性光感受器，其光循环与感官视紫红质相似。在此，我们报告了pyp基因的克隆和测序，以及发色团及其与蛋白质的共价连接的化学鉴定。将蛋白水解衍生的发色肽的高分辨率质谱元素组成数据、蛋白质结合和化学释放的发色团的pH滴定和紫外可见光谱，以及游离发色团酰胺的碎片质谱与1.4 Å分辨率的蛋白质晶体结构结果相结合，以确定PYP中的发色团为通过硫酯键与唯一的半胱氨酸残基共价结合的4-羟基肉桂基。虽然4-羟基肉桂酸是苯丙烷途径的代谢产物和植物应激反应中的关键分子，但这是该基团对蛋白质进行共价修饰的首次报道。在PYP的黑暗（黄色）状态下，蛋白质将发色团稳定为去质子化的酚盐阴离子。通过将我们对发色团的生化表征与其他已发表的观察结果相结合，我们提出了光循环的化学基础：在最初吸收光子后，PYP的光循环涉及发色团质子化形成对应于观察到的光漂白中间体的中性酚形式。
