Differential scanning fluorimetry measurement of protein stability changes upon binding to glycosaminoglycans: a screening test for binding specificity.
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.
Uniewicz
Katarzyna A
KA
School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, United Kingdom.
Ori
Alessandro
A
Xu
Ruoyan
R
Ahmed
Yassir
Y
Wilkinson
Mark C
MC
Fernig
David G
DG
Yates
Edwin A
EA
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
D006025:Glycosaminoglycans; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1