15N NMR Relaxation Studies of Free and Ligand-bound Human Acidic Fibroblast Growth Factor

• Published in: The Journal of biological chemistry 2000-12, Vol.275 (50), p.39444-39450
• Main Authors: Chi, Ya-hui, Kumar, Thallampuranam Krishnaswamy S., Chiu, Ing-Ming, Yu, Chin
• Format: Article
• Language: English
• Subjects: Binding Sites; Electrophoresis, Polyacrylamide Gel; Escherichia coli - metabolism; Fibroblast Growth Factor 1 - chemistry; Fibroblast Growth Factor 1 - metabolism; Humans; Isomerism; Ligands; Magnetic Resonance Spectroscopy; Models, Chemical; Protein Binding; Protein Conformation; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M007205200

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 steady-state 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.