title: 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.
authors.affiliation: School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, United Kingdom.
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Uniewicz
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ori
authors.forename: Ruoyan
authors.initials: R
authors.lastname: Xu
authors.forename: Yassir
authors.initials: Y
authors.lastname: Ahmed
authors.forename: Mark C
authors.initials: MC
authors.lastname: Wilkinson
authors.forename: David G
authors.initials: DG
authors.lastname: Fernig
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Yates
mesh_terms: D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D006025:Glycosaminoglycans; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1