Facile Method for High-throughput Identification of Stabilizing Mutations
Christensen, Signe LU ; Wernersson, Camille and André, Ingemar LU
(2023)
In Journal of Molecular Biology
435(18).
- Abstract
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. However, 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 sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of... (More)
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. However, 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 sorting, 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 °C increase in thermal 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.
(Less)
- author
- Christensen, Signe
LU
; Wernersson, Camille
and André, Ingemar
LU
- organization
- publishing date
- 2023-09-15
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- deep mutational scanning, folding reporter, high-throughput screening, multiplex assay, thermostability
- in
- Journal of Molecular Biology
- volume
- 435
- issue
- 18
- article number
- 168209
- publisher
- Elsevier
- external identifiers
-
- pmid:37479080
- scopus:85166229420
- ISSN
- 0022-2836
- DOI
- 10.1016/j.jmb.2023.168209
- language
- English
- LU publication?
- yes
- id
- c4529943-d9af-493f-96d1-447c207220f7
- date added to LUP
- 2023-10-23 15:30:43
- date last changed
- 2024-04-19 02:46:53
@article{c4529943-d9af-493f-96d1-447c207220f7,
abstract = {{<p>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. However, 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 sorting, 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 °C increase in thermal 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.</p>}},
author = {{Christensen, Signe and Wernersson, Camille and André, Ingemar}},
issn = {{0022-2836}},
keywords = {{deep mutational scanning; folding reporter; high-throughput screening; multiplex assay; thermostability}},
language = {{eng}},
month = {{09}},
number = {{18}},
publisher = {{Elsevier}},
series = {{Journal of Molecular Biology}},
title = {{Facile Method for High-throughput Identification of Stabilizing Mutations}},
url = {{http://dx.doi.org/10.1016/j.jmb.2023.168209}},
doi = {{10.1016/j.jmb.2023.168209}},
volume = {{435}},
year = {{2023}},
}