Issue 4, 2020

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 by 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.

Graphical abstract: Long-range PEG stapling: macrocyclization for increased protein conformational stability and resistance to proteolysis

Supplementary files

Article information

Article type
Paper
Submitted
19 Мам. 2020
Accepted
03 Там. 2020
First published
13 Там. 2020
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2020,1, 273-280

Long-range PEG stapling: macrocyclization for increased protein conformational stability and resistance to proteolysis

Q. Xiao, D. S. Ashton, Z. B. Jones, K. P. Thompson and J. L. Price, RSC Chem. Biol., 2020, 1, 273 DOI: 10.1039/D0CB00075B

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements