The Structural and Functional Properties of a Double Mutant of Human Acidic Fibroblast Growth Factor (hFGF-1)
Date of Graduation
Master of Science in Cell & Molecular Biology (MS)
Suresh K. Thallapuranam
Second Committee Member
Human acidic Fibroblast Growth Factor 1 (FGF-1), a member of the FGF superfamily, is a potent mitogen and heparin-binding protein involved in a broad spectrum of biological processes, including angiogenesis, cell proliferation, and wound healing. Design of hFGF-1 with an increased thermal stability and an enhanced cell proliferation activity is highly desired for wound healing applications. Herein, we have designed the variant of FGF-1 by substituting two important amino residues in the heparin-binding pocket. The variant was overexpressed in Escherichia coli and was successfully purified to homogeneity using an affinity chromatographic procedure. Far-UV circular dichroism spectroscopic analysis showed that the backbone conformation of the hFGF-1 did not alter due to the introduction of mutations in the heparin-binding pocket. The designed hFGF-1 variant exhibited an increased resistance to limited trypsin digestion. Isothermal titration calorimetry study confirmed that approximately 20-fold decrease in heparin binding affinity (Kd ~90µM) was observed in case of the double mutant compared to that of the wild-type FGF1 (~5µM). Incorporation of positively charged Lys135 adjacent to the negatively charged E136 might have reduced the repulsive effect to heparin. 8-Anilino naphthalene 1-sulfonate (ANS) binding assay revealed that the introduced mutations cause a subtle change in the solvent-accessible non-polar surface of the protein. These results were in concordant with other biophysical data obtained from limited trypsin digestion, 1H 15N HSQC analysis. In addition, no significant change in bioactivity was observed between the mutant and the wild-type FGF1 proteins. This confirms that introduction of positive charge adjacent to E136 nullified the effects of this unique mutation.
Alghanmi, A. M. (2017). The Structural and Functional Properties of a Double Mutant of Human Acidic Fibroblast Growth Factor (hFGF-1). Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/2481