title: Thermodynamic characterization of mutants of human fibroblast growth factor 1 with an increased physiological half-life.
abstract: 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.
authors.affiliation: Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee 32306-4380, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Culajay
authors.forename: S I
authors.initials: SI
authors.lastname: Blaber
authors.forename: A
authors.initials: A
authors.lastname: Khurana
authors.forename: M
authors.initials: M
authors.lastname: Blaber
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D019791:Guanidine; D006207:Half-Life; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D012694:Serine / Q000235:genetics; D013816:Thermodynamics
chemical_list: D016220:Fibroblast Growth Factor 1; D012694:Serine; D019791:Guanidine; D003545:Cysteine