This proposal describes investigations aimed at determining the role of stability and irreversible denaturation in the regulation of human acidic fibroblast growth factor (haFGF), a growth factor involved in normal embryonic development, wound healing, angiogenesis, as well as in the maintenance of some solid tumors. Like many regulatory proteins, haFGF has a short half life in vivo, which, in this case, appears to be due to generally poor thermal stability, which operates in conjunction with structural mechanisms (i.e., buried free cysteine residues) to lead to irreversible denaturation. This proposal is aimed generally at investigating protein stability as a regulatory mechanism that may be common to the regulation of short lived potent signaling proteins. A combination of methodologies will be used. Hypotheses about the structural basis of stability will be tested by the introduction of specific changes in the sequence of haFGF. X-ray crystallography will be used to determine the structural effects of these mutations. High sensitivity differential scanning calorimetry will be used to determine the effects of mutations upon thermodynamic parameters of unfolding, and upon reversible folding characteristics. Cell assays involving the stimulation of DNA synthesis in fibroblasts will be used to determine the effects of mutation upon function. Three regions of the protein are selected for mutation, based upon hypotheses of the potential origins of the protein's instability. Internal cysteine residues will be substituted by alanine to determine whether they contribute to instability, solely by promoting the irreversible phase of denaturation via disulfide formation. Lysines that form the basic cluster involved in heparin binding will be mutated to determine the role of charge repulsion within this cluster, in the absence of bound anions or heparin, in decreasing stability. Mutations will be introduced into residues surrounding the protein's central cavity, to reduce the size of this cavity, and ligands will be introduced into the central cavity, to determine the role of the cavity in protein instability.
| Blaber, S I; Culajay, J F; Khurana, A et al. (1999) Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride. Biophys J 77:470-7 |
