Abstract
In this study, the thermodynamic and backbone dynamics properties of human acidic fibroblast growth factor (hFGF-1) are described. hFGF-1 is a ~ 16 kDa, all b-barrel protein which plays key roles in several important cellular processes related to morphogenesis, development and angiogenesis. The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional 1H-15N HSQC spectra of hFGF-1 acquired at sub zero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. To describe the internal motions of hFGF-1 ranging from pico- to millisecond, 15N NMR relaxation data have been used to characterize the backbone dynamics of hFGF-1 in its free and sucrose octasulfate (SOS) bound states. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, the conformational exchange behavior of hFGF-1 is tremendously reduced upon SOS binding. Also, the receptor-binding segment comprising residues 103-111 shows increased flexibility in the presence of SOS. To investigate the internal motions of hFGF-1 range from millisecond to days, hydrogen-deuterium exchange experiments in the absence and low concentrations of denaturant were performed. In contrast to the equilibrium unfolding events monitored by optical probes, native-like state hydrogen exchange data shows that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII and IX exchange by the sub-folding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. The work described here elucidates the correlation between the structural dynamics with biological function of hFGF-1. This work also proves that the slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.