title: Dimerization capacities of FGF2 purified with or without heparin-affinity chromatography.
abstract: Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.
authors.affiliation: INSERM U1029, Allée Geoffroy St. Hilaire, Pessac, France; Université Bordeaux I, Allée Geoffroy St. Hilaire, Pessac, France.
authors.forename: Natalia
authors.initials: N
authors.lastname: Platonova
authors.affiliation: INSERM U1029, Allée Geoffroy St. Hilaire, Pessac, France; Université Bordeaux I, Allée Geoffroy St. Hilaire, Pessac, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Miquel
authors.affiliation: Institute of Bioinformatics and Structure Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Liang-Yuan
authors.initials: LY
authors.lastname: Chiu
authors.affiliation: INSERM U1053, Team Avenir, Bordeaux, France.
authors.forename: Said
authors.initials: S
authors.lastname: Taouji
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Milano, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Moroni
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Milano, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Colombo
authors.affiliation: INSERM U1053, Team Avenir, Bordeaux, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Chevet
authors.affiliation: Institute of Bioinformatics and Structure Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Shih-Che
authors.initials: SC
authors.lastname: Sue
authors.affiliation: INSERM U1029, Allée Geoffroy St. Hilaire, Pessac, France; Université Bordeaux I, Allée Geoffroy St. Hilaire, Pessac, France.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bikfalvi
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D055503:Protein Multimerization; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry / Q000378:metabolism
chemical_list: D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D006493:Heparin