Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system.
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.
Moreau
Morgane J J
MJ
Comparative Genomics Centre, School of Pharmacy & Molecular Sciences, James Cook University, DB 21, James Cook Drive, Townsville, QLD 4811, Australia.
Morin
Isabelle
I
Schaeffer
Patrick M
PM
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
D008024:Ligands; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins