Probing the affinity of polyanions for acidic fibroblast growth factor by unfolding kinetics.
The relationship between ligand-protein affinity and the extent of protein stabilization induced by such interactions has been investigated using the binding of polyanions to acidic fibroblast growth factor (aFGF) as a model system. It was found that the experimentally observed unfolding rate constant of aFGF consists of two components: one equal to the unfolding rate constant of the aFGF-ligand complex and the other the product of the unfolding rate constant of free aFGF, the aFGF-ligand dissociation constant (Kd), and the reciprocal of the molar ligand concentration. This reflects the presence of two possible unfolding pathways: at high ligand excess dissociation is suppressed and slow unfolding of the aFGF-ligand complex itself prevails. When lower concentrations of ligand allows equilibrium-driven appearance of free aFGF, a more rapid unfolding of dissociated protein predominates. Existence of a steady state of dissociated aFGF undergoing unfolding was demonstrated by computer simulation of the elementary events, using experimentally determined rate constants. The potential applications of such simulations are outlined. An equation allowing estimation of dissociation constants from equilibrium denaturation curves obtained in the presence of a varying amount of ligand is also proposed. In addition, determination of initial unfolding rates in the presence of excess protein permits the the stoichiometry of the interaction to be determined.
Mach
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Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Middaugh
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D003198:Computer Simulation; D004926:Escherichia coli; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D000071228:Polyelectrolytes; D011108:Polymers / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
D016220:Fibroblast Growth Factor 1; D000071228:Polyelectrolytes; D011108:Polymers; D011994:Recombinant Proteins