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

摘要

The interaction between glycosaminoglycans (GAGs) andnproteins is important for the regulation of protein trans-nport and activity. Here we present a novel method for thenmeasurement of protein GAG interactions suitable fornhigh-throughput screening, able to discriminate betweennthe interactions of a protein with GAGs of differentnstructures. Binding of proteins to the GAG heparin, anproxy for sulfated regions of extracellular heparan sulfate,nwas found to enhance the stability of three test proteins,nfibroblast growth factors (FGFs)-1, -2, and -18. Chemi-ncally modified heparins and heparin oligosaccharides ofndifferent lengths stabilized the three FGFs to differentnextents, depending on the pattern of sugar binding speci-nficity. The method is based on a differential scanningnfluorescence approach. It uses a Sypro Orange dye, whichnbinds to exposed core residues of a denatured protein andnresults in an increased fluorescence signal. It is convenient,nrequiring low micromolar amounts of protein and ligandncompared to other interaction assays, employing only a real-ntime polymerase chain reaction (PCR) instrument.