ArticleProtein science : a publication of the Protein Society2022
Saturation mutagenesis of a predicted ancestral Syk-family kinase.
Article in Protein science : a publication of the Protein Society, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 13 citations in OpenAlex.
- LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation.Proceedings of the National Academy of Sciences of the United States of America · 2026Article
- Context-dependent peptide recognition shapes tyrosine kinase substrate specificity beyond consensus motifs.bioRxiv : the preprint server for biology · 2026Article
- Uncovering the molecular basis of kinase activity and substrate recognition with phospho-PCA.bioRxiv : the preprint server for biology · 2025Article
- Allosteric modulation of NF1 GAP: Differential distributions of catalytically competent populations in loss-of-function and gain-of-function mutants.Protein science : a publication of the Protein Society · 2025Article
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- Ancestral Reconstruction and the Evolution of Protein Energy Landscapes.Annual review of biophysics · 2024Review
- Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.bioRxiv : the preprint server for biology · 2024Article
- Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function.The Journal of biological chemistry · 2024Review
- ProteinGym: Large-Scale Benchmarks for Protein Design and Fitness Prediction.bioRxiv : the preprint server for biology · 2023Article
- ProteinNPT: Improving Protein Property Prediction and Design with Non-Parametric Transformers.bioRxiv : the preprint server for biology · 2023Article
- Article
- Saturation mutagenesis of a predicted ancestral Syk-family kinase.Protein science : a publication of the Protein Society · 2022Article
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Authors and funding
6 authors at 5 institutions in 1 country.
Funding
Abstract
Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.
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