title: Solubilization of hydrophobic peptides by reversible cysteine PEGylation.
abstract: "PEG-a-Cys" reagent, synthesized by the esterification of monomethoxy-poly(ethylene glycol) (avg. MW = 5 kDa) to Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], is shown to "PEGylate" reversibly the cysteine residue of a 25-residue synthetic hydrophobic peptide (H2N-REAAALAAAAALAAWAALCPARRRR-CO2H) designed to model a transmembrane segment of a membrane protein. A mixed disulfide bond was formed between the reagent and the peptide that was readily cleaved with the mild reducing agent tricarboxyethylphosphine hydrochloride (TCEP.HCl). Carboxypeptidase B digestion of the charged carboxyl terminus of the peptide through to the Ala residue--which mimics the enzymatic cleavage of a TM segment from a fusion protein--releases a highly hydrophobic peptide. A time-dependent decrease in the amplitude of the digested peptide circular dichroism (CD) spectra was attributed to the aggregation and/or precipitation of the peptide. While PEGylation of the peptide with PEG-a-Cys had a negligible effect on conformation, it inhibited the loss of CD amplitude in both intact and digested peptides, suggesting that it was effective in solubilization of hydrophobic peptides.
authors.affiliation: Division of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: N C
authors.initials: NC
authors.lastname: Pomroy
authors.forename: C M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D000595:Amino Acid Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010720:Phosphines / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D012995:Solubility
chemical_list: D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D003545:Cysteine; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D010455:Peptides; D010720:Phosphines; D011092:Polyethylene Glycols; C080938:tris(2-carboxyethyl)phosphine