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Long-range PEG Stapling: Macrocyclization for Increased Protein Conformational Stability and Resistance to
Qiang Xiao1, Dallin S Ashton1, Zachary B Jones1
1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.
Protein stapling with PEG oligomers enhances conformational stability. This method improves protein folding and resistance to degradation, applicable to various protein types like WW and SH3 domains.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Engineering
Background:
- Protein conformational stability is crucial for function and therapeutic applications.
- PEGylation is a common strategy to modify protein properties.
- Previous work demonstrated long-range PEG stapling enhances WW domain stability via an entropic effect.
Purpose of the Study:
- To validate criteria for identifying effective PEG-stapling sites in β-sheet proteins (WW and SH3 domains).
- To compare the efficacy of olefin metathesis and copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) for protein stapling.
- To assess the impact of protein stapling on proteolytic resistance and quaternary structure stability.
Main Methods:
- PEGylation of Asn-linked O-allyl PEG oligomers.
- Olefin metathesis and CuAAC for creating PEG staples.
- Circular dichroism spectroscopy and differential scanning fluorimetry to measure conformational stability.
- Limited proteolysis assays to evaluate proteolytic resistance.
- Analysis of an α-helical GCN4 coiled-coil heterodimer for quaternary structure stability.
Main Results:
- Stapling criteria were validated for WW and SH3 domains, confirming that distant primary sequence sites close in tertiary structure yield optimal stability.
- Both olefin metathesis and CuAAC provided similar energetic benefits, indicating interchangeability of olefin and triazole staples.
- PEG-stapled proteins exhibited increased resistance to proteolysis.
- An intermolecular staple significantly enhanced the quaternary structural stability of a GCN4 coiled-coil heterodimer.
Conclusions:
- Long-range PEG stapling is a versatile strategy to enhance protein conformational and proteolytic stability in β-sheet proteins.
- The choice between olefin and triazole staples does not significantly impact energetic benefits.
- Protein engineering via stapling offers a promising approach for developing more robust protein therapeutics and biomaterials.
- Intermolecular stapling can stabilize protein-protein interactions and quaternary structures.
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