Facile Method for High-throughput Identification of Stabilizing Mutations

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. How-ever, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sort-ing, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 & DEG;C increase in ther-mal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.& COPY; 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom-mons.org/licenses/by/4.0/).