Date of Graduation

5-2022

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Thallapuranam, Suresh

Committee Member/Reader

Lessner, Daniel

Committee Member/Second Reader

Walker, James

Committee Member/Third Reader

Chapman, Kate M.

Abstract

Fibroblast growth factors are a family of heparin binding proteins that are involved in a broad spectrum of physiological functions, such as angiogenesis, cell proliferation, cell differentiation, and wound healing. Fibroblast growth factor 1 (FGF-1) is a member of the FGF superfamily and can bind universally to fibroblast growth factor receptors. Fibroblast growth factor 2 (FGF-2) is similar in structure to FGF-1 but has been shown to have a potent effect on angiogenesis. A physiologically stable dimerization of these two growth factors could prove to induce desirable effects in the instance of wound healing. In this study, we have designed a FGF1/2 heterodimer composed of FGF-1 and FGF-2 linked together by a glycine linker. We introduced a charge reversal mutation (R136E) within the heparin binding pocket of FGF-1 component of the heterodimer. The variant was expressed using E. coli and purified using heparin Sepharose column chromatography. Circular dichroism showed the variant did not alter the backbone confirmation and fluorescence spectroscopy indicated that the tertiary structure of the variant remained intact as well. An increase in resistance to trypsin digestion, thermal denaturation, and urea denaturation was seen in the variant in comparison to the wild-type FGF1/2 protein. This confirms that the introduction of R136E induced overall stability when in comparison to the wild-type. This mutation acts a starting point for the FGF1/2 heterodimer. Future studies will explore the resistance to thrombin and proliferative effects of this mutation as well as examine the mutation in the presence of additional stabilizing mutations.

Keywords

Fibroblast Growth Factors; FGF-1; FGF-2; characterization; heterodimer

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