COMPLETE CHEMICAL-STRUCTURE OF PHOTOACTIVE YELLOW PROTEIN - NOVEL THIOESTER-LINKED 4-HYDROXYCINNAMYL CHROMOPHORE AND PHOTOCYCLE CHEMIST
COMPLETE CHEMICAL-STRUCTURE OF PHOTOACTIVE YELLOW PROTEIN - NOVEL THIOESTER-LINKED 4-HYDROXYCINNAMYL CHROMOPHORE AND PHOTOCYCLE CHEMIST
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DOI：
10.1021/bi00252a001
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发表时间：
1994-12-06
期刊：
影响因子：
2.9
通讯作者：
GETZOFF, ED
中科院分区：
文献类型：
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作者：
BACA, M;BORGSTAHL, GEO;GETZOFF, ED
光活性蛋白捕获和利用光子能量的独特能力取决于发色团、其与蛋白质的连接以及周围的蛋白质环境。为了了解发色团和蛋白质相互作用以经历光周期的分子机制，我们正在研究光敏黄色蛋白（PYP），这是一种来自嗜盐外硫红螺菌的 14 kDa 水溶性光感受器，其光周期与感觉视紫红质相似。在这里，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、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、以及释放的发色团酰胺的碎片质谱的元素组成数据与 1.4 埃分辨率的蛋白质晶体结构的结果相结合，将 PYP 中的发色团鉴定为共价结合的 4-羟基肉桂基。通过硫酯键连接的唯一半胱氨酸残基。虽然 4-羟基肉桂酸酯是苯丙素途径的代谢产物，也是植物胁迫反应中的关键分子，但这是该组对蛋白质进行共价修饰的第一份报告。在 PYP 的暗（黄色）状态下，该蛋白质以去质子化的酚阴离子的形式稳定发色团。通过将我们对生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：在光子的初始吸收之后，PYP的光循环涉及生色团的质子化为与观察到的光漂白中间体相对应的中性苯酚形式。
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 W-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-Angstrom-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.