Automated selection of stabilizing mutations in designed and natural proteins
Open Access
- 17 January 2012
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 109 (5), 1494-1499
- https://doi.org/10.1073/pnas.1115172109
Abstract
The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins.Keywords
This publication has 27 references indexed in Scilit:
- Rosetta3Methods in Enzymology, 2010
- RosettaHoles2: A volumetric packing measure for protein structure refinement and validationProtein Science, 2010
- Practically Useful: What the RosettaProtein Modeling Suite Can Do for YouBiochemistry, 2010
- RosettaHoles: Rapid assessment of protein core packing for structure prediction, refinement, design, and validationProtein Science, 2008
- Protein production by auto-induction in high-density shaking culturesProtein Expression and Purification, 2005
- An Orientation-dependent Hydrogen Bonding Potential Improves Prediction of Specificity and Structure for Proteins and Protein–Protein ComplexesJournal of Molecular Biology, 2003
- Extending the accuracy limits of prediction for side-chain conformationsJournal of Molecular Biology, 2001
- Tertiary templates for proteinsJournal of Molecular Biology, 1987
- The interpretation of protein structures: Estimation of static accessibilityJournal of Molecular Biology, 1971
- A Coefficient of Agreement for Nominal ScalesEducational and Psychological Measurement, 1960