GMMA Can Stabilize Proteins Across Different Functional Constraints.

Autor: Daffern N; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80305, USA., Johansson KE; Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark., Baumer ZT; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80305, USA., Robertson NR; Department of Bioengineering, University of California, Riverside, USA., Woojuh J; Department of Botany and Plant Sciences, University of California, Riverside, USA., Bedewitz MA; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80305, USA., Davis Z; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80305, USA., Wheeldon I; Department of Chemical and Environmental Engineering, University of California, Riverside, USA; Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA, USA., Cutler SR; Department of Botany and Plant Sciences, University of California, Riverside, USA; Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA, USA; Center for Plant Cell Biology, University of California, Riverside, Riverside, CA, USA., Lindorff-Larsen K; Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark. Electronic address: lindorff@bio.ku.dk., Whitehead TA; Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80305, USA. Electronic address: timothy.whitehead@colorado.edu.
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 2024 Jun 01; Vol. 436 (11), pp. 168586. Date of Electronic Publication: 2024 Apr 23.
DOI: 10.1016/j.jmb.2024.168586
Abstrakt: 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.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KL-L holds stock options in and is a consultant for Peptone Ltd. T.A.W. is on the scientific advisory board at Metaphore Biotechnologies. The remaining authors declare no competing interests.
(Copyright © 2024 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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