| Summary: | 博士 === 國立清華大學 === 化學系 === 92 === Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes. The biological activities of FGFs are mediated by cell surface receptors or FGFRs. We have expressed the FGF-binding (or D2) domain of FGFR in Escherichia coli in high yields (10 mg /liter) and recovered the D2 protein by dissolving in 8 M urea, subsequently refolding on the nickel affinity column and purification using heparin-sepharose affinity resin. Far UV circular dichroism data and 1Hα, 13Cα, 13Cβ and 13CO chemical shift indices suggested that the D2 domain is an all beta-sheet protein consisting of 9 beta-strands. Isothermal titration calorimetry and equilibrium urea unfolding experiments showed that the recombinant D2 domain was in a biologically active conformation and binds strongly to its ligand FGF and to the heparin analog, sucrose octasulfate (SOS).
Using a variety of triple resonance NMR experiments, assignments of the 1H, 15N and 13C resonances in the D2 domain have been completely accomplished. The three-dimensional structure of the D2 domain was calculated using distance-geometry followed by simulated annealing techniques with ARIA-CNS. RDC restraints were also incorporated for structure refinements. 15N T1, T2 and steady-state NOE values at two magnetic fields (500 MHz and 800 MHz) further defined regions of the D2 domain in rigid structure formation as well as with flexible surface loops.
The binding regions of hFGF-1 and SOS on the isolated D2 protein were characterized using 1H-15N HSQC perturbation experiments and compared these specific ligand-binding interactions with the related crystal complex structures. A peptide spanning residues 11 to 28 of the isolated D2 domain sequence was synthesized to identify the specific ligand-binding interactions between the D2 domain and hFGF-1. Size exclusion fast performance liquid chromatography (FPLC) provides a direct approach to monitor the association/ dissociation state of the D2 domain with/ without SOS and hFGF-1 molecules.
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