Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.

Human acidic fibroblast growth factor (FGF-1) is a potent mitogen and angiogenic factor, with reportedly poor thermal stability and a relatively short in vivo half-life. However, certain mutants of FGF-1 have been described that exhibit a significant increase in half-life in tissue culture-based assays. FGF-1 contains three cysteine residues, two of which are highly conserved and buried within the protein core. Mutant forms of FGF-1 that substitute a serine residue at these cysteine positions have been reported to increase the protein's half-life and specific activity as well as decrease the dependence upon heparin for full activity. However, the underlying physical basis for this increase in half-life has not been determined. Possible effects include stabilization of protein structure and elimination of sulfhydryl chemistry at these positions. Here we have used differential scanning calorimetry and isothermal equilibrium denaturation to characterize thermodynamic parameters of unfolding for individual, and combination, cysteine to serine mutations in human FGF-1. The results show that substitution by serine is destabilizing at each cysteine position in wild-type FGF-1. Thus, the increased half-life previously reported for these mutations does not correlate with thermal stability and is most likely due to elimination of sulfhydryl chemistry. The results also suggest a method by which protein half-life may be modulated by rational design.