15N NMR relaxation studies of free and ligand-bound human acidic fibroblast growth factor

Abstract

15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. 15N longitudinal (R1), transverse (R2) relaxation rates and {1H}-15N steadystate nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 °C) for all resolved backbone amide groups using 1H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (τm) for the free and SOS-bound forms were estimated to be 10.4 ± 1.07 and 11.1 ± 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant Rex values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization, hFGF-1 is a rigid molecule with an average generalized parameter (S2) value of 0.89± 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S2 = 0.94 ± 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103-111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein.