Solubilization of hydrophobic peptides by reversible cysteine PEGylation

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 tricarboxlyethylphosphine 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, (C) 1998 Academic Press.