Abstract
Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2–6 °C in thermal inactivation temperature and no decrease in functionality.
Originalsprog | Engelsk |
---|---|
Artikelnummer | 168586 |
Tidsskrift | Journal of Molecular Biology |
Vol/bind | 436 |
Udgave nummer | 11 |
Antal sider | 15 |
ISSN | 0022-2836 |
DOI | |
Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:We thank Prof. Jakob R. Winther for discussions and insights related to GMMA. This work was supported by Defense Advanced Research Projects Agency Advanced Plant Technologies (DARPA APT, HR001118C0137) to T.A.W. S.R.C. and I.W; National Science Foundation GRFP to Z.T.B; the PRISM (Protein Interactions and Stability in Medicine and Genomics) centre funded by the Novo Nordisk Foundation (NNF18OC0033950) to K.L.L. K.E.J. and K.L.L acknowledge access to computational resources at the Biocomputing Core Facility at the Department of Biology, University of Copenhagen. The views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. Approved for Public Release, Distribution Unlimited.
Funding Information:
We thank Prof. Jakob R. Winther for discussions and insights related to GMMA. This work was supported by Defense Advanced Research Projects Agency Advanced Plant Technologies (DARPA APT, HR001118C0137) to T.A.W., S.R.C., and I.W; National Science Foundation GRFP to Z.T.B; the PRISM (Protein Interactions and Stability in Medicine and Genomics) centre funded by the Novo Nordisk Foundation (NNF18OC0033950) to K.L.L. K.E.J. and K.L.L acknowledge access to computational resources at the Biocomputing Core Facility at the Department of Biology, University of Copenhagen. The views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. Approved for Public Release, Distribution Unlimited.
Publisher Copyright:
© 2024 Elsevier Ltd