Ligand screening using fluorescence thermal shift analysis (FTS).

Area of Science:

• Biophysics
• Structural Genomics
• Drug Discovery

Background:

• The fluorescence thermal shift (FTS) is a biophysical technique monitoring protein unfolding via temperature-induced changes.
• It relies on hydrophobic probes that fluoresce upon binding to exposed surfaces during protein denaturation.
• FTS is valuable for assessing protein thermal stability and identifying interacting molecules.

Purpose of the Study:

• To detail the principles, protocols, and data analysis for FTS screening within the Center for Structural Genomics of Infectious Diseases (CSGID) pipeline.
• To present FTS as a high-throughput method adaptable for both robotic and manual assay formats.
• To demonstrate the utility of FTS in drug discovery screening and protein function studies.

Main Methods:

• Utilizing a fluorescence-based assay to detect protein unfolding as a function of temperature.
• Employing hydrophobic fluorescent probes to monitor changes in protein surface exposure.
• Implementing a high-throughput 384-well format adaptable to a 96-well manual setup.

Main Results:

• Detailed protocols for FTS screening applicable to structural genomics pipelines.
• Demonstrated adaptability of the FTS assay for various throughput needs.
• Successful application of FTS in screening 100 proteins against a chemical library.

Conclusions:

• FTS is a versatile and efficient technique for structural genomics and drug discovery.
• The described protocols facilitate high-throughput screening and analysis of protein thermal stability.
• FTS provides a robust platform for identifying ligands and characterizing protein function.