PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.

The interaction of a positively charged amino acid residue with a negatively charged residue (i.e. a salt bridge) can contribute substantially to protein conformational stability, especially when two ionic groups are in close proximity. At longer distances, this stabilizing effect tends to drop off precipitously. However, several lines of evidence suggest that salt-bridge interaction could persist at longer distances if an aromatic amino acid residue were positioned between the anion and cation. Here we explore this possibility in the context of a peptide in which a Lys residue occupies the i + 8 position relative to an i-position Glu on the solvent-exposed surface of a helix-bundle homotrimer. Variable temperature circular dichroism (CD) experiments indicate that an i + 4-position Trp enables a favorable long-range interaction between Glu and the i + 8 Lys. A substantial portion of this effect relies on the presence of a hydrogen-bond donor on the arene; however, non-polar arenes, a cyclic hydrocarbon, and an acyclic Leu side-chain can also enhance the long-range salt bridge, possibly by excluding water and ions from the space between Glu and Lys.