Thermal stability & kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity & stability can be separately designed

Dylan Alexander Carlin, Siena Hapig-Ward, Bill Wayne Chan, Natalie Damrau, Mary Riley, Ryan W. Caster, Bowen Bethards, Justin Siegel

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat, KM,) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat, KM, in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.

Original languageEnglish (US)
Article numbere0176255
JournalPLoS One
Volume12
Issue number5
DOIs
StatePublished - May 1 2017

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Glycoside Hydrolases
Enzyme activity
hydrolases
thermal stability
glycosides
Thermodynamic stability
Hot Temperature
enzyme activity
kinetics
Protein Engineering
Enzyme Stability
Kinetics
Protein Stability
Enzymes
protein engineering
enzyme stability
Proteins
prediction
Escherichia coli
Mutation

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Thermal stability & kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity & stability can be separately designed. / Carlin, Dylan Alexander; Hapig-Ward, Siena; Chan, Bill Wayne; Damrau, Natalie; Riley, Mary; Caster, Ryan W.; Bethards, Bowen; Siegel, Justin.

In: PLoS One, Vol. 12, No. 5, e0176255, 01.05.2017.

Research output: Contribution to journalArticle

Carlin, Dylan Alexander ; Hapig-Ward, Siena ; Chan, Bill Wayne ; Damrau, Natalie ; Riley, Mary ; Caster, Ryan W. ; Bethards, Bowen ; Siegel, Justin. / Thermal stability & kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity & stability can be separately designed. In: PLoS One. 2017 ; Vol. 12, No. 5.
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