Cys(i)-Lys(i+3)-Lys(i+4) triad: a general approach for PEG-based stabilization of α-helical proteins.

PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs. Modern chemoselective reactions now enable specific placement of a single PEG at any site on a protein surface. However, few rational structure-based guidelines exist for selecting optimal PEGylation sites. Here, we explore the impact of PEGylation on the conformational stability of alpha-helices using an alpha-helical coiled coil as a model system. We find that maleimide-based PEGylation of a solvent-exposed i position Cys can stabilize coiled-coil quaternary structure when Lys residues occupy both the i + 3 and i + 4 positions, due to favorable interactions between the PEG-maleimide and the Lys residues. Applying this Cys(i)-Lys(i+3)-Lys(i+4) triad to a solvent-exposed position within the C-terminal helix of the villin headpiece domain leads to similar PEG-based increases in conformational stability, highlighting the possibility of using the Cys(i)-Lys(i+3)-Lys(i+4) triad as a general strategy for PEG-based stabilization of helical proteins.