Engineering the Structure of the Human Acidic Fibroblast Growth Factor to Enhance its Stability and Cell Proliferation Activity

Human acidic fibroblast growth factor (hFGF1) is a protein well known for its role in cell growth and differentiation. To elicit these cell-signaling processes, hFGF1 non-selectively binds to any one of the seven cell surface hFGF receptor isoforms. Due to its significant involvement in tissue repair activity, hFGF1 is a prime candidate for novel wound healing therapeutics. However, one drawback toward its use as a novel wound healing therapeutic is the poor inherent thermal stability of hFGF1, as it has been found to unfold near physiological temperature. The cause of this instability is strong electrostatic repulsion created by a dense cluster of positively charged amino acids near the c-terminus. This instability leads to proteolytic degradation of the unfolded protein, which severely limits the bioavailability of hFGF1. To counteract this instability, hFGF1 binds with high affinity to the heavily sulfated glycosaminoglycan, heparin, which eliminates the charge-charge repulsion via electrostatic interactions with the positively charged residues near the c-terminus in the region known as the heparin-binding pocket. However, recently several disadvantages have been acknowledged with the use of heparin in hFGF1 wound-healing therapeutics. Thus, to address these issues, we have genetically engineered several rationally designed point mutations within and near by the heparin-binding region of hFGF1 to modulate the heparin-binding affinity and to increase the thermal stability and cell proliferation activity of the protein. Study of each mutation is performed with biophysical experiments as well as molecular dynamics simulations (which are found as supplementary files)