Impact of Site-Specific PEGylation on the Conformational Stability and Folding Rate of the Pin WW Domain Depends Strongly on PEG Oligomer Length

Abstract

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.

Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies

“…At its most advanced stage, conjugate research applies the tools of medicinal chemistry by incorporating insights from structure–function studies into the design of the molecule. A study 64 of the site-specific pegylated WW domain of the human protein peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) 65 showed that conformational stability depends on the molecular mass of PEG; longer oligomers increased the folding rate and reduced the rate of protein unfolding.…”

How PEGylation influences protein conformational stability

“…We recently showed that attaching a short PEG (comprising four ethylene oxide units) to a single Asn side chain at position 19 in the WW domain of the human protein Pin 1 increases WW conformational stability by −0.70 ± 0.04 kcal mol −1 due to accelerated folding and slowed unfolding [57] (see figure 2). Shorter PEG chains impart less stability to WW than the four-unit PEG, whereas longer PEG chains provide similar stability.…”

Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate

“…The difference in stability between 1-nbp and 1p cannot be attributed solely to the fact that 1-nbp contains four more ethylene oxide units than 1p : branched 1-nbp is also more stable than a previously characterized derivative of 1p that has eight ethylene oxide units instead of four. 25 This observation highlights the possibility that branched PEGs may be more effective at increasing protein stability than linear PEGs of similar molecular weight.…”