title: Ligand screening using fluorescence thermal shift analysis (FTS).
abstract: The fluorescence thermal shift (FTS) method is a biophysical technique that can improve productivity in a structural genomics pipeline and provide a fast and easy platform for identifying ligands in protein function or drug discovery screening. The technique has gained widespread popularity in recent years due to its broad-scale applicability, throughput, and functional relevance. FTS is based on the principle that a protein unfolds at a critical temperature that depends upon its intrinsic stability. A probe that will fluoresce when bound to hydrophobic surfaces is used to monitor protein unfolding as temperature is increased. In this manner, conditions or small molecules that affect the thermal stability of a protein can be identified. Herein, principles, protocols, data analysis, and special considerations of FTS screening as performed for the Center for Structural Genomics of Infectious Diseases (CSGID) pipeline are described in detail. The CSGID FTS screen is designed as a high-throughput 384-well assay to be performed on a robotic platform; however, all protocols can be adapted to a 96-well format that can be assembled manually. Data analysis can be performed using a simple curve fitting of the fluorescent signal using a Boltzmann or double Boltzmann equation. A case study of 100 proteins screened against Emerald Biosystem's ADDit™ library is included as discussion.
authors.affiliation: High-Throughput Analysis Laboratory, Department of Molecular Biosciences, Center for Structural Genomics of Infectious Diseases, Northwestern University, Evanston, IL, 60208, USA.
authors.forename: Chi-Hao
authors.initials: CH
authors.lastname: Luan
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Light
authors.forename: Sara F
authors.initials: SF
authors.lastname: Dunne
authors.forename: Wayne F
authors.initials: WF
authors.lastname: Anderson
mesh_terms: D003141:Communicable Diseases / Q000235:genetics*; D023281:Genomics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D008967:Molecular Biology / Q000379:methods*; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*
chemical_list: D008024:Ligands; D011506:Proteins