Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.
Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, United States.
ACS Chem Biol. 2022 May 20;17(5):1015-1021. doi: 10.1021/acschembio.2c00013. Epub 2022 Apr 28.
Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.
由于表达疏水性跨膜（TM）结构域具有挑战性，因此将功能性 TM 蛋白重构到模型膜中具有挑战性，这些 TM 结构域通常需要稳定的去污剂，而这些去污剂可能会破坏蛋白质的结构和功能。最近的模型系统通过将膜蛋白的可溶性结构域与脂质连接来解决这个问题，使用的是非共价共轭。在此，我们测试了一种替代方法，涉及使用化学选择性肽键合将合成的 TM 结构域和表达的可溶性结构域组装成 TM 蛋白。我们开发了一种内含肽介导的连接策略，通过将可溶性蛋白结构域共价连接到合成的 TM 多肽上，在合成的巨大单层囊泡（GUV）膜中半合成单次跨膜蛋白，从而避免了去污剂的使用。我们表明，程序性细胞死亡蛋白 1 的细胞外结构域在与 GUV 中的合成 TM 肽连接后保留了其在膜界面处与配体结合的功能，从而促进了受体-配体相互作用的研究。
