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.
References
Jun 5, 1992·Journal of Molecular Biology·E M MeieringA R Fersht
Jan 17, 1995·Proceedings of the National Academy of Sciences of the United States of America·B K ShoichetB W Matthews
Aug 6, 1996·Biochemistry·L P McIntoshS G Withers
Jun 7, 2002·IUBMB Life·Thomas K Harris, George J Turner
Jun 25, 2002·Journal of Molecular Biology·Raphael GueroisLuis Serrano
Jul 30, 2002·Journal of Molecular Biology·Beth M Beadle, Brian K Shoichet
Jun 28, 2005·Nucleic Acids Research·Joost SchymkowitzLuis Serrano
Dec 31, 2005·Nucleic Acids Research·M D Shaji KumarAkinori Sarai
Apr 4, 2006·Proceedings of the National Academy of Sciences of the United States of America·Jesse D BloomFrances H Arnold
Jun 26, 2007·Journal of Molecular Biology·Pablo IsornaJulia Sanz-Aparicio
Mar 21, 2008·Nature·Daniela RöthlisbergerDavid Baker
May 9, 2008·PLoS Computational Biology·Nobuhiko TokurikiDan S Tawfik
Jun 17, 2009·Proceedings of the National Academy of Sciences of the United States of America·Jesse D Bloom, Frances H Arnold
Nov 17, 2009·Journal of Molecular Biology·Veena L ThomasBrian K Shoichet
Nov 26, 2009·Nature Reviews. Molecular Cell Biology·Philip A Romero, Frances H Arnold
Jul 17, 2010·Nature Reviews. Genetics·Misha Soskine, Dan S Tawfik
Jul 22, 2010·Science·Justin B SiegelDavid Baker
Feb 3, 2011·Proteins·Elizabeth H KelloggDavid Baker
May 24, 2011·PloS One·Florian RichterDavid Baker
May 29, 2012·Nature Biotechnology·Timothy A WhiteheadDavid Baker
Feb 21, 2013·Methods in Enzymology·Andrew Leaver-FayBrian Kuhlman
Dec 17, 2014·Chemical Society Reviews·Andrew CurrinDouglas B Kell
Jun 4, 2015·Proceedings of the National Academy of Sciences of the United States of America·Philip A RomeroAdam R Abate
Sep 17, 2015·Journal of the American Chemical Society·Clancey WolfIngrid S Pultz
Jan 28, 2016·PloS One·Dylan Alexander CarlinJustin B Siegel
Mar 10, 2016·Nature Communications·Jan-Ytzen van der MeerGerrit J Poelarends
Jul 19, 2016·Molecular Cell·Adi GoldenzweigSarel J Fleishman
Nov 26, 2016·Current Opinion in Structural Biology·Emily E WrenbeckTimothy A Whitehead
Citations
Jun 8, 2021·Biophysical Chemistry·Marius Sobamfou KambiréLucien Patrice Kouamé
Jul 3, 2021·Frontiers in Bioengineering and Biotechnology·Marc SchererElena Bencurova
Apr 8, 2020·ACS Omega·Peishan HuangJustin B Siegel
Dec 14, 2017·Biochemistry·Srivatsan Raman