Faculty of Life Science, Institute of Biochemistry, Leipzig University, Leipzig 04103, Germany.
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065, United States.
Chem Rev. 2024 Nov 27;124(22):12498-12550. doi: 10.1021/acs.chemrev.4c00181. Epub 2024 Nov 7.
Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free "click" chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.
基于化学生物学的方法为整体膜蛋白的研究做出了重大贡献。最近的一个关键方法是遗传密码扩展（GCE）的应用，它可以实现将具有特定化学性质的非天然氨基酸（ncAAs）定点掺入蛋白质中。高效的 GCE 具有挑战性，特别是对于具有专门生物发生和细胞运输机制的膜蛋白，它们在细胞膜中的表达水平通常较低。许多真核膜蛋白无法在 中有效表达，在哺乳动物细胞培养系统中研究它们最为有效。最近的进展促进了 GCE 在膜蛋白研究中的更广泛应用。首先，已经设计了 AARS/tRNA 对，以在哺乳动物细胞中高效发挥作用。其次，生物正交化学反应，包括细胞友好的无铜“点击”化学，使得可以将小分子探针（如荧光团）与活细胞中的膜蛋白连接起来。最后，与 GCE 方法学的进步相协调，可用的 ncAAs 的种类急剧增加，从而能够通过多学科的生化和生物物理方法研究蛋白质结构和动力学。本文在 GCE 技术发展的历史框架内对这些进展进行了综述，重点介绍了它们在膜蛋白研究中的应用。
