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Long-range PEG Stapling: Macrocyclization for Increased Protein Conformational Stability and Resistance to Proteolysis.

ABSTRACT: We previously showed that long-range stapling of two Asn-linked O-allyl PEG oligomers via olefin metathesis substantially increases the conformational stability of the WW domain through an entropic effect. The impact of stapling was more favorable when the staple connected positions that were far apart in primary sequence but close in the folded tertiary structure. Here we validate these criteria for identifying new stabilizing PEG-stapling sites within the WW domain and the SH3 domain, both β-sheet proteins. We find that stapling via olefin metathesis vs. the copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) results in similar energetic benefits, suggesting that olefin and triazole staples can be used interchangeably. Proteolysis assays of selected WW variants reveal that the observed staple-based increases in conformational stability lead to enhanced proteolytic resistance. Finally, we find that an intermolecular staple dramatically increases the quaternary structural stability of an α-helical GCN4 coiled-coil heterodimer.

SUBMITTER: Xiao Q

PROVIDER: S-EPMC8009319 | biostudies-literature |

REPOSITORIES: biostudies-literature

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