Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States.
Biochemistry. 2022 Jul 19;61(14):1495-1507. doi: 10.1021/acs.biochem.2c00118. Epub 2022 Jun 23.
Understanding the structure and structure-function relationships of membrane proteins is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins , we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional inner nuclear membrane Sad1/UNC-84 (SUN) proteins (i.e., SUN1 and SUN2) in supported lipid bilayers. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and heterotypic interactions between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins . While our platform is not a substitute for cell-based studies, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.
理解膜蛋白的结构和结构-功能关系是生物医学研究中的一个基本问题。鉴于对膜蛋白进行机械生化和生物物理研究固有的困难，我们之前开发了一种简便的基于 HeLa 细胞的无细胞表达（CFE）系统，该系统能够有效地在支持的脂质双层中重新构建全长（FL）功能性核内膜 Sad1/UNC-84（SUN）蛋白（即 SUN1 和 SUN2）。在这里，我们提供的证据表明，在支持的脂质双层中重新构建 CFE 合成的 FL 膜蛋白主要通过存在于我们的 CFE 反应中的内质网衍生的微体与我们的支持的脂质双层融合来发生。此外，我们证明了我们的合成生物学平台很容易用于研究化学环境对 CFE 合成的 FL SUN 蛋白在支持的脂质双层中与 KASH 蛋白 nesprin-2 的腔域相互作用的能力的影响。此外，我们使用该平台研究了 SUN1 和 SUN2 之间同型和异型相互作用的分子要求。最后，我们表明我们的平台可用于在单个支持的脂质双层中同时重新构建三种不同的 CFE 合成的 FL 膜蛋白。总体而言，这些结果确立了我们基于 HeLa 细胞的 CFE 和支持的脂质双层重建平台作为对 FL 膜蛋白的寡聚化和功能进行机制剖析的有力工具。虽然我们的平台不能替代基于细胞的研究，但它确实为难以研究的膜蛋白的生物学提供了重要的机制见解。
