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.15 N longitudinal (R 1 ), transverse (R 2 ) relaxation rates and { 1 H}-15 N steadystate nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25°C) for all resolved backbone amide groups using 1 H-detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time ( m ) 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 (R ex ) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant R ex 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 (S 2 ) value of 0.89 ؎ 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S 2 ‫؍‬ 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.

14-3-3ζ C-terminal Stretch Changes Its Conformation upon Ligand Binding and Phosphorylation at Thr232

“…This observation somewhat contradicts the general assumption that ligand binding reduces motion of the protein (39,40). However, in several cases ligand-induced increase in the protein flexibility has been observed (41)(42)(43). For example, it has been shown that the backbone motion of the mouse major urinary binding protein I increases upon binding of the pheromone 2-secbutyl-4,5-dihydrothiazole (41).…”

“…For example, it has been shown that the backbone motion of the mouse major urinary binding protein I increases upon binding of the pheromone 2-secbutyl-4,5-dihydrothiazole (41). Studies of the backbone dynamics of the human acidic fibroblast growth factor revealed that while many residues show decrease in the mobility, a segment comprising nine residues became more flexible in the presence of the ligand sucrose octasulfate (42). Moreover the 14-3-3 C-terminal stretch is not a part of the ligand binding groove but rather the adjacent segment, which can probably occupy the binding groove and thus has to be ejected from the groove during ligand binding (6).…”

Amyloid-like Fibril Formation in an All β-Barrel Protein Involves the Formation of Partially Structured Intermediate(s)

“…In the present study, we demonstrate that even a ␤-barrel protein such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1) 1 forms amyloid-like fibrils (23)(24)(25)(26). Furthermore, by using a variety of biophysical techniques we show that amyloid-like fibril formation in nFGF-1 involves the formation of a partially structured intermediate state (s).…”

Characterization of the Structure and Dynamics of a Near-native Equilibrium Intermediate in the Unfolding Pathway of an All β-Barrel Protein

“…The backbone dynamics of hFGF-1 in the native state has been investigated previously (58). In this study, we were particularly interested in understanding the trends in the transverse relaxation rates (R 2 ) (in the native and intermediate states), as they are sensitive to the dynamics on the microsecond-millisecond time scale.…”