Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1
Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1
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DOI：
10.1073/pnas.0831227100
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发表时间：
2003-04-29
影响因子：
11.1
通讯作者：
Engelhard, M
中科院分区：
文献类型：
--
作者：
Becker, CFW;Hunter, CL;Engelhard, M
通过化学方法产生生物功能对于理解和靶向生理过程具有潜在的重要性。蛋白质的化学合成提供了以定制的方式改变靶蛋白质分子的性质的能力。在目前的工作中，它表明，这种方法可以扩展到阐明蛋白质-蛋白质相互作用的例子，由完整的化学合成的原癌基因产物H-Ras以及其效应c-Raf 1的Ras结合结构域（RBD）。H-Ras的166-aa多肽链通过三个未保护的肽段的天然化学连接来合成。类似地，通过连接两个肽区段来制备81-aa RBD。这两个RBD和Ras显示的功能和光谱特性无法区分，从他们的重组形式的判断CD光谱和瞬态动力学测量的Ras-RBD相互作用，以及从核苷酸取代反应在Ras。将带有硝基苯并呋咱侧链的非天然氨基酸引入RBD的91位，提供独特的荧光性质。用Ras-鸟苷5 '-β，γ-亚氨三磷酸标记的硝基苯并呋咱的缔合瞬态显示出在早期工作中通过使用其他信号未检测到的缓慢相。
Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein-protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras-RBD interaction as well as from nucleotide replacement reactions in Ras. An unnatural amino acid bearing a nitrobenzofurazan side chain was introduced into position 91 of the RBD, providing unique fluorescence properties. The association transient of nitrobenzofurazan labeled with Ras-guanosine 5'-beta,gamma-imidotriphosphate showed a slow phase that had not been detected in earlier work by using other signals.