title: Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system.
abstract: Information about the stability of proteins is paramount to determine their optimal storage or reaction conditions. It is also essential to determine protein stability in high-throughput when screening for new or improved functions of proteins obtained from large mutant libraries. In drug discovery programs, monitoring of ligand-induced stabilization effects can be used to identify lead compounds in high-throughput. These studies require expensive biophysical instrumentation and large quantities of purified proteins. To address these issues, we developed a new method, using GFP as a reporter system to quantify the stability of a protein and its ligand-associated stabilization effects that requires neither special equipment nor extensive purification steps. Here, GFP is fused to a protein of interest (POI) through a linker and is used as a reporter system for protein unfolding and aggregation. The three POIs used in this study include the Ter-binding protein Tus, glycerol kinase and chloramphenicol acetyl transferase. The fluorescent fusion protein is subjected to irreversible thermal denaturation leading to formation of aggregates, which are eliminated by a centrifugation step. The residual fluorescence of the soluble fraction can be directly related to the stability of the POI and can be quantitatively monitored using a fluorescence plate reader. The GFP-based stability assay (GFP-Basta) was able to identify stabilizing compounds and afforded a new quantitative method for the screening and ranking of ligands for three different proteins. These applications are particularly useful for drug discovery, directed evolution, structural and functional genomics.
authors.affiliation: Comparative Genomics Centre, School of Pharmacy & Molecular Sciences, James Cook University, DB 21, James Cook Drive, Townsville, QLD 4811, Australia.
authors.forename: Morgane J J
authors.initials: MJ
authors.lastname: Moreau
authors.forename: Isabelle
authors.initials: I
authors.lastname: Morin
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
mesh_terms: D000465:Algorithms; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D055550:Protein Stability; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D008024:Ligands; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins