Ahmed, S and Manjunath, K and Chattopadhyay, G and Varadarajan, R (2022) Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries. In: Journal of Biological Chemistry, 298 (4).
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Abstract
Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, especially for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site saturation mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, saturation suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addition, multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temperature of about 20 0 C, and that displayed enhanced tolerance to high temperature exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants. © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
| Item Type: | Journal Article |
|---|---|
| Publication: | Journal of Biological Chemistry |
| Publisher: | American Society for Biochemistry and Molecular Biology Inc. |
| Additional Information: | The copyright for this article belongs to author |
| Keywords: | Mutagenesis; Proteins; Screening, Fluorescence activated cell sorting; Improved activities; Ligand binding; Mutant libraries; Point mutations; Protein library; Protein stability; Protein structure prediction; Saturation mutagenesis; Single sites, Libraries |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 17 May 2022 06:49 |
| Last Modified: | 17 May 2022 06:49 |
| URI: | https://eprints.iisc.ac.in/id/eprint/71761 |
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