Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins

Moreau, Morgane J.J., Morin, Isabelle, Askin, Samuel P., Cooper, Alanna, Moreland, Nicole J., Vasudevan, Subhash G., and Schaeffer, Patrick M. (2012) Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. RSC Advances, 2 (31). pp. 11892-11900.

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Abstract

The development of differential scanning fluorimetry and the high-throughput capability of Thermofluor have vastly facilitated the screening of crystallization conditions of proteins and large mutant libraries in structural genomics programs, as well as ligands in drug discovery and functional genomics programs. These techniques are limited by their requirement for both highly purified proteins and solvatochromic dyes, fueling the need for more robust technologies that can be used with crude protein samples. Here, we present the development of a new high-throughput technology for the quantitative determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. This technology is based on the principle that a change in the proximal environment of GFP, such as unfolding and aggregation of the protein of interest, is measurable through its effect on the fluorescence of the fluorophore. Protein stability data was generated for twelve GFP-tagged proteins including monomeric and multimeric, DNA-binding, RNA-binding, proteolytic, heat-shock and metabolic proteins of Escherichia coli, Burkholderia pseudomallei, Staphylococcus aureus, dengue and influenza (H5N1) viruses. The technology is simple, fast and insensitive to variations in sample volumes, and the useful temperature and pH range is 30–80 °C and 5–11 respectively. The system does not require solvatochromic dyes, reducing the risk of interference. The protein samples are simply mixed with the test conditions in a 96-well plate and subjected to a melt-curve protocol using a real-time thermal cycler. The data are obtained within 1–2 h and include unique quality control measures.

Item ID: 25658
Item Type: Article (Refereed Research - C1)
ISSN: 2046-2069
Date Deposited: 19 Mar 2013 04:56
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060109 Proteomics and Intermolecular Interactions (excl Medical Proteomics) @ 40%
06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 20%
06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060107 Enzymes @ 40%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970103 Expanding Knowledge in the Chemical Sciences @ 20%
97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 40%
97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 40%
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