Figure 1.
Chemical shift perturbation upon variation in FGF2 concentrations.
A. HSQC spectra of heparin column purified FGF2 of 1.5 mM (purple), 1.1 mM (Blue), 0.55 mM (green), 0.275 mM (orange) and 0.137 mM (red). B. HSQC spectra of ion-exchange column purified FGF2 of 1 mM (black) and 0.125 mM (red). Chemical shift difference is only observed in the heparin-column purified FGF2 and the difference is analyzed by the combined chemical shift perturbation, [(ΔδNH2 + ΔδN2/25)/2]1/2. C. Chemical shift perturbation of the heparin-column purified FGF2 is between 0.137 mM and 0.55 mM and (D) the residues with perturbation larger than the threshold value of 0.01 ppm were highlighted on the FGF2 structure (PDB code: 1BLA) with yellow spheres. E. Chemical shift perturbation of the heparin-column purified FGF2 between 0.137 mM and 1.5 mM and (F) the residues with perturbations larger than the threshold values of 0.012, 0.015 and 0.02 ppm were pointed out FGF2 structure as yellow, orange and red spheres, respectively where the radius of sphere reflects the quantity of chemical shift perturbation. The proposed two dimer interfaces are indicated by dash lines.
Figure 2.
Comparison of HSQC spectra purified by two different methods.
(A) Superposition of HSQC spectra of heparin-affinity purified (blue) and ion-exchange purified FGF2 (red). (B) Chemical shift differences between the two spectra analyzed by the combined chemical shift perturbation are indicated.
Figure 3.
Chemical shift perturbation upon changing FGF2 concentrations in the presence of heparin tetrasaccharide (hep-4).
(A) HSQC spectra of FGF2 in complex with hep-4 with molar ratio of 1: 0.5. FGF2 at 0.125 mM (black) and 1.0 mM (red). The inset shows the close view of resonances of G47 and G51. (B) The corresponding close view of HSQC spectra of heparin column purified FGF2: 0.137 mM (black) and 1.5 mM (red). (C) Chemical shift perturbation of FGF2/hep-4 complex for FGF2 at 0.125 mM and 1.0 mM. (D) The residues in FGF2 with perturbations larger than the threshold values of 0.01 ppm are indicated as yellow spheres.
Figure 4.
FGF2 dimerization in C-N orientation.
A. Dose-dependent proliferation of blood endothelial cells induced by recombinant FGF2-C-His and FGF2-N-GST as well as commercial FGF2. Proliferation of cells grown for 72 h was determined by Cell Proliferation Reagent WST-1. One representative experiment is shown, values are as the mean ± SEM (n = 4). In the inset, recombinant FGF2-C-His and FGF2-N-GST were analyzed by 10% denaturating SDS-PAGE. Protein levels were visualized by Coomassie staining. Molecular weight marker is indicated on the left. B. Detection of FGF2 dimerization by AlphaScreen™. Top panel: assay design of the AlphaScreen™ experiment. FGF2-N-GST was bound to AlphaScreen™ Glutathione Donor beads and FGF2-C-His to AlphaScreen™ Ni chelate acceptor beads. Bottom panel: Direct interaction between FGF2-N-GST and FGF2-C-His. The recombinant proteins were incubated at indicated concentrations with donor and acceptor beads for 24 h at room temperature before signal measurements. Histograms are representative of three independent experiments with comparable results. Data are expressed as mean ± SEM (n = 4). C. Cross-linking assay of FGF2. 1µg FGF2 labeled with near-infrared fluorescent IRDye800CW (LICOR Biosciences) was incubated with or without the cross-linker BS3. Cross-linking samples were analyzed by 10% SDS-PAGE under reducing conditions. The IR signal was visualized using Odyssey Infrared Imaging System (LICOR Biosciences). The arrows show cross-linked FGF2 oligomers. Molecular weight marker is indicated on the right. D. A solid-phase ligand-binding assay with immobilized 500 nM FGF2-C-His and 50 nM soluble FGF2-N-GST in the presence or absence of 1µg/ml heparin. The dimerization was revealed as described in the Material and Methods. Representative experiment was done in duplicate. Error bars represent the mean ±SEM (n = 4).
Figure 5.
Competition assay to assess FGF2 dimerization (C-N orientation) using the AlphaScreen™ technology.
A. Competition assay of FGF2 dimerization with heparin-like substances. The interaction between 50 nM FGF2-N-GST and 50 nM FGF2-C-His was competed in the presence of increasing concentrations of heparin. Tag less FGF2 (B) and PF4 (C) compete for FGF2 dimerization of 50 nM FGF2-N-GST and 50 nM FGF2-C-His. D. Anionic citrate inhibits heparin-dependent and independent FGF2 dimerization. (A through D) AlphaScreen™ signal is expressed as percentages in comparison of control; AlphaScreen™ signal corresponding to FGF2 dimer without competitors is set as 100%. Results are representative of at least three independent experiments. Results are mean values ± SEM (n = 3).
Figure 6.
FGF2 dimerization and competition in the N-N orientation.
A. Top panel: assay design of the AlphaScreen™ experiment. FGF2-C-His was bound to AlphaScreen™ Ni chelate donor and acceptor beads. Bottom panel: For dimerization, increasing amounts of FGF2-C-His were incubated with the beads for 24 h before AlphaScreen™ signal measurements. B. Competition assay of FGF2-C-His dimerization at various concentrations of FGF2 with increasing concentrations of heparin (left). FGF2 dimer formation in the presence of 250 ng/ml heparin (right). FGF2-GST (C) and PF4 (D) compete for 1µM FGF2-C-His dimerization. (A through D) Results are representative of at least three independent experiments. Results are mean values ± SEM (n = 3).