| Autor: |
Podolsky KA; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States., Masubuchi T; Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, United States., Debelouchina GT; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States., Hui E; Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, United States., Devaraj NK; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States. |
| Abstrakt: |
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 in vitro 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. |
