Novel thioester reagents afford efficient and specific S-acylation of unprotected peptides under mild conditions in aqueous solution.
S-acylated peptides have many potential uses for elucidating the biophysical, structural and other properties of the numerous S-acylated proteins of mammalian cells. However, with the currently available reagents, preparation of specifically S-acylated derivatives of peptides is generally laborious or simply unfeasible. We here show that novel, easily preparable aryl and alkyl thioester derivatives of palmitic acid can mediate S-acylation of peptides corresponding to physiologically S-acylated sequences from the proteins p56(lck) and H-ras and the Po glycoprotein of peripheral myelin, with high selectivity for cysteine over other amino acid functional groups (including hydroxyl and both alpha- and epsilon-amino residues), and with much greater efficiency than is obtained using acyl-coenzyme A derivatives. Efficient and selective S-acylation can be accomplished under very mild conditions in aqueous systems containing lipid vesicles or detergent micelles, or in homogenous aqueous/acetonitrile mixtures. Using these novel thioesterifying reagents, we confirm previous suggestions that the N-terminal cysteine residue of Hedgehog proteins can exhibit rapid, uncatalyzed S-to-N acyl transfer following S-acylation to produce the N-palmitoylated amino terminus found in the mature protein. By contrast, we demonstrate that spontaneous S-to-N acyl transfer from the cysteine to the terminal glycine residue in the amino-terminal peptide of G(alphas) is far less rapid and is likely too slow to explain the physiological N-palmitoylation of the amino terminus of this protein.
Sang
S Leung Wai
SL
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Silvius
J R
JR
D000215:Acylation; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D053823:Hedgehog Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry; D018631:ras Proteins / Q000737:chemistry
D044385:GTP-Binding Protein alpha Subunits; C099256:Galpha5 protein, Dictyostelium; D006023:Glycoproteins; D053823:Hedgehog Proteins; D010455:Peptides; D015800:Protozoan Proteins; D018631:ras Proteins