A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide. Attachment of lipophilic groups is an important post‐translational modification of proteins. To study its impact on the membrane partitioning of hydrophobic peptides/proteins, we designed a tyrosine‐based trifunctional linker that allows facile incorporation of two different functionalities at a cysteine residue in a single step. Shown is a liquid‐disordered phase of a giant unilamellar vesicle (green semicircle), trifunctional linker (blue) connected to a fluorophore (black), DPPE lipid (brown), and protein.