Date of Graduation

5-2022

Document Type

UAF Access Only - Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Thallapuranam, Suresh

Committee Member/Reader

Du, Yuchun

Committee Member/Second Reader

Ivey, Mack

Committee Member/Third Reader

Plavcan, Joseph M.

Abstract

Fibroblast growth factors (FGFs) are a family of heparin-binding proteins involved in important biological processes such as cell proliferation, angiogenesis, tumor growth, and wound healing. FGF proteins are often used for tissue regeneration, but are subject to thermal, chemical, and proteolytic denaturation causing loss of function before they can be utilized. A novel FGF1-FGF2 heterodimer was designed which can exhibit the cell proliferation properties of FGF1 and the angiogenesis properties of FGF2. Based on previous work from the Kumar Lab, three mutations (R136E, K126N, and Q54P) were created on the FGF1 subunit of the heterodimer to stabilize the heparin-binding pocket (HBP) of the molecule. The HBP is a cluster of positively charged residues located at the C-terminal end (residues K126 to Q141) of FGF1 which facilitates heparin binding. Studies have shown that inherent instability of FGF1 is largely contributed by the presence of the HBP. Introduction of negatively charged amino acids into the HBP through site directed mutagenesis proved to increase the stability of the protein in various studies. The R136E and K126N mutations replace positively charged arginine and lysine by negatively charged glutamate and polar asparagine in the HBP. This contributes to added stability in proven studies. The R136E mutation also eliminates susceptibility of FGF1 to thrombin digestion by altering a secondary cleavage site for thrombin. Q54P, the third mutation, creates a more compact molecule, serving to increase stability of the protein. Large-scale expression, purification, and characterization experiments were performed on the wildtype, R136E (single mutant), R136E/K126N (double mutant), and R136E/K126N/Q54P (triple mutant) FGF1-FGF2 heterodimers to compare their thermal, chemical, and proteolytic stability. The triple mutant heterodimer exhibited the greatest overall stability in the presence and absence of heparin when compared to the other variants.

Keywords

proteins; FGF; biochemistry

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