Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon Binding to Glycosaminoglycans: A Screening Test for Binding Specificity

Abstract

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

“…The well documented successes in expediting protein stabilization, drug discovery (especially in less well financed laboratories) and crystallization 10,[23][24][25] have made it an attractive method for initial screening of compounds. Compounds added to proteins show a clear dose dependent increase in the apparent melting temperature 7,9 .…”

The sirtuin SIRT6 regulates stress granules formation in C. elegans and in mammals

“…Again, and typical for this dye-binding assay, thermal unfolding was irreversible (Phillips and de la Peña, 2011). Relative to changes in the fluorescence characteristics of tryptophan residues, differential scanning fluorimetry is able to detect the exposure of hydrophobic patches during the unfolding of the protein core (Uniewicz et al, 2010). It is thus conceivable that the midpoint of this process is reached at later stages of thermal unfolding relative to the solvent exposure of the tryptophan residues.…”

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions

“…Recombinant FGF Purification-pET-14b-FGF-1, pET-14b-FGF-2, pETM-11-FGF-9, pETM-11-FGF-18, and pETM-11-FGF-21 were transformed into Escherichia coli C41 (DE3) cells and expressed, as described previously (15)(16)(17). pETM-11-FGF-7 was transformed into E. coli BL21 (DE3) cells, expressed by 1 mM isopropyl 1-thio-␤-D-galactopyranoside at 37°C for 3 h, and purified as described for FGF-18 (15).…”