Zheng Ji-Shen, He Yao, Zuo Chao, Cai Xiao-Ying, Tang Shan, Wang Zhipeng A, Zhang Long-Hua, Tian Chang-Lin, Liu Lei
High Magnetic Field Laboratory, Chinese Academy of Sciences, and School of Life Sciences, University of Science and Technology of China , Hefei 230031, China.
Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University , Beijing 100084, China.
J Am Chem Soc. 2016 Mar 16;138(10):3553-61. doi: 10.1021/jacs.6b00515. Epub 2016 Mar 4.
Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies.
化学蛋白质合成能够提供具有翻译后修饰或位点特异性标记的蛋白质。尽管这项技术在水溶性球状蛋白的研究中应用越来越广泛，但膜蛋白的化学合成仍然难以实现。在本报告中，开发了一种通用且稳健的可去除主链修饰（RBM）方法用于膜蛋白的化学合成。该方法在Fmoc固相肽合成过程中使用一种活化的O-到-N酰基转移辅助剂来安装一个对三氟乙酸具有可切换反应性的RBM基团。由于RBM基团可以置于任何一级氨基酸上，所以该方法可应用于多种膜蛋白。有了RBM，膜蛋白及其片段的行为几乎就如同它们是水溶性肽一样，并且在连接、纯化和质量表征过程中易于处理。全长蛋白组装完成后，RBM基团可以很容易地通过三氟乙酸去除。通过成功合成具有位点特异性同位素标记的双跨膜结构域蛋白（丙型肝炎病毒p7离子通道）和四跨膜结构域蛋白（多药耐药转运蛋白EmrE），证明了该新方法的效率和实用性。此方法能够实际合成用于生化和生物物理研究的中小型膜蛋白或膜蛋白结构域。
