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Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon Binding to Glycosaminoglycans: A Screening Test for Binding Specificity
journal contributionposted on 2010-05-01, 00:00 authored by Katarzyna A. Uniewicz, Alessandro Ori, Ruoyan Xu, Yassir Ahmed, Mark C. Wilkinson, David G. Fernig, Edwin A. Yates
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.
denatured proteinheparin oligosaccharidesfibroblast growth factorsFGFDifferential Scanning Fluorimetry MeasurementSypro Orange dyeinteraction assaysPCRprotein transportcore residuesfluorescence signalScreening Testsugar binding specificityProtein Stability Changessulfated regionspolymerase chain reactionGAG heparinBinding SpecificityThe interactiontest proteinsmicromolar amountsextracellular heparan sulfatescanning fluorescence approachnovel method