GMMA Can Stabilize Proteins Across Different Functional Constraints

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
Article number168586
JournalJournal of Molecular Biology
Volume436
Issue number11
Number of pages15
ISSN0022-2836
DOIs
Publication statusPublished - 2024

Bibliographical note

Publisher Copyright:
© 2024 Elsevier Ltd

    Research areas

  • Computational Design, Deep Sequencing, GMMA, Protein Engineering, Protein Thermostability

ID: 391677846