Alkylation of cysteine-containing peptides to mimic palmitoylation

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function prop erly. The molecular details by which this palmitoyl modification affects pr otein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C-16 groups a t cysteine residues and demonstrated using peptides derived from the palmit oylated region of Sindbis virus E2 glycoprotein. The synthetic approach tak es advantage of disulfide chemistry to specifically modify only the cystein e residues within peptides and covalently links C-16 groups via disulfide b ridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The ch emistry presented here takes place in solution under mild conditions withou t the need for protection of the peptide functional groups. A method for pu rifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biologi cal systems.