Improving Split Reporters of Protein-Protein Interactions through Orthology-Based Protein Engineering

ACS Chem Biol. 2024 Feb 16;19(2):428-441. doi: 10.1021/acschembio.3c00631. Epub 2024 Jan 30.

Abstract

Protein-protein interactions (PPIs) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of splitFAST2, an improved reporter displaying higher brightness, lower self-complementation, and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, splitFAST2 is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Protein Engineering
  • Protein Interaction Mapping*
  • Proteins* / genetics
  • Proteins* / metabolism

Substances

  • Proteins