PEGylation near a Patch of Nonpolar Surface Residues Increases the Conformational Stability of the WW Domain

Abstract

Many proteins have one or more surface-exposed patches of nonpolar residues; our observations here suggest that PEGylation near such locations might be a useful strategy for increasing protein conformational stability. Specifically, we show that conjugating a PEG-azide to a propargyloxyphenylalanine via the copper­(I)-catalyzed azide–alkyne cycloaddition can increase the conformational stability of the WW domain due to a favorable synergistic effect that depends on the hydrophobicity of a nearby patch of nonpolar surface residues.

Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity

“…In this case, the optimal increase in proteolytic stability was linked with a conformational stability high increase. Furthermore, they found the (identity dependent linker between PEG and protein) alternative PEGylation strategies which effectively alters the WW protein conformational stability [22]. The location, length of PEG, and chemistry used 3 BioMed Research International to connect PEG with proteins may influence conformation stability.…”

“…PEGylation at the N-terminal did not substantially change the stability of SH3 [ 21 ]. Residue specific PEGylation was shown to strengthen the Glu12-Arg14 salt-bridge by shielding it from the interference of water molecules [ 22 ]. Recently, Zuma and colleagues (2022) recently demonstrated that site-specific PEGylation enhances the biological activity and stability of recombinant DNA ligase proteins [ 23 ].…”

“…Draper et al [ 22 ] recently reported that specific modification of Asn residue on the side chain amide nitrogen within the WW domain with a 190 kDa monomethoxyPEG greatly enhanced WW conformational and proteolytic stability. In this case, the optimal increase in proteolytic stability was linked with a conformational stability high increase.…”

Molecular Dynamics Simulations for Rationalizing Polymer Bioconjugation Strategies: Challenges, Recent Developments, and Future Opportunities

“…Rational design guidelines are needed to drive forward such optimizations , and pave the way for viable, biocompatible PEG alternatives. − Therefore, a thorough understanding of the underlying protein–polymer structure and its dynamics is crucial. Unfortunately, harnessing the full potential of the typical experimental approaches used in structural biology, such as X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance (NMR) spectroscopy, is inherently difficult for bioconjugates with their highly flexible polymer chains. − Experimental investigations into protein–polymer dynamics mainly rely on stability data obtained from systematic mutation studies, − deducing structure–function–dynamics relationships indirectly. Alternative techniques have been reported, such as hydrogen–deuterium exchange experiments, small-angle neutron or X-ray scattering (SANS/SAXS), ,− spin labeling of polymers for subsequent NMR measurements and limited-proteolysis (LiP) assays …”