doi:10.1016/j.molcel.2009.11.022
Optimizing Protein Stability In Vivo
Linda Foit1, 6, 7, Gareth J. Morgan3, 4, 7, Maximilian J. Kern1, Lenz R. Steimer1, Annekathrin A. von Hacht1, James Titchmarsh3, 5, Stuart L. Warriner3, 5, Sheena E. Radford3, 4, , and James C.A. Bardwell1, 2, ,
1 Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109, USA
2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
3 Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
4 Institute for Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK
5 School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
6 Institute for Chemistry and Pharmacy, University of Münster, Münster 48149, Germany
Corresponding author
Corresponding author
7 These authors contributed equally to this work
Summary
Linda Foit1, 6, 7, Gareth J. Morgan3, 4, 7, Maximilian J. Kern1, Lenz R. Steimer1, Annekathrin A. von Hacht1, James Titchmarsh3, 5, Stuart L. Warriner3, 5, Sheena E. Radford3, 4, , and James C.A. Bardwell1, 2, ,
1 Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109, USA
2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
3 Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
4 Institute for Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK
5 School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
6 Institute for Chemistry and Pharmacy, University of Münster, Münster 48149, Germany
Corresponding author
Corresponding author
7 These authors contributed equally to this work
Summary
Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of proteins to antibiotic resistance. It allows the identification of stabilizing mutations within proteins. The large majority of mutants selected for improved antibiotic resistance are stabilized both thermodynamically and kinetically, indicating that similar principles govern stability in vivo and in vitro. The approach requires no prior structural or functional knowledge and allows selection for stability without a need to maintain function. Mutations that enhance thermodynamic stability of the protein Im7 map overwhelmingly to surface residues involved in binding to colicin E7, showing how the evolutionary pressures that drive Im7-E7 complex formation have compromised the stability of the isolated Im7 protein.
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