Date of Graduation

12-2016

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Thallapuranam, Suresh

Committee Member

Tian, Z. Ryan

Second Committee Member

Henry, Ralph L.

Keywords

FGF1; FGFR; Heparin; Wound healing

Abstract

Fibroblast growth factor receptor (FGFR) is made up of three significant domains. The most important domain is the intracellular domain where the dimerization and autophosphorylation occur. Fibroblast growth factor (FGF) interacts with specific FGFR to regulate many cellular processes during the embryonic stage. Furthermore, FGF is significant for adults because FGF plays an important role in regulating cellular differentiation as well as wound healing. The cellular regulating processes are initiated through binding FGF to heparin followed by binding FGF/heparin to FGFR to form FGF/heparin/FGFR complex. Thus, FGFR is dimerized and autophosphorylated. The phosphorylation of FGFR triggers downstream signaling pathways, which enhance cellular processes. Any destruction of downstream signaling results in several diseases including different types of cancer. FGF1, which consists of 140 or 154 amino acids, is made of 12 β-antiparallel strands. Over the past twenty years, several mutations were engineered in various sites to enhance protein stability and function. In our work, penta mutations Q40P, S47L, H93S, K112N, and R122E were introduced in FGF1 to increase protein stability and enhance its biological activity. Because pFGF1 lost heparin binding ability, pFGF1 was not purified by heparin as WFGF1. The pFGF1 was purified by nickel-sepharose column because of inserting poly His tag in its N-terminal. Based on NMR and fluorescence results, the tertiary structure of pFGF1 is different from WFGF1. The pFGF1 exhibits more stability than WFGF1 because thermodynamic study shows that pFGF1 rises the denaturation temperature by ~20 ºC. Also, urea denaturation study illustrates that pFGF1 is partially unfolded after being exposed to 4M urea. Moreover, pFGF1 is not affected by trypsin because it shows high resistance to proteolytic enzymes even if the time of incubation is increased. Despite the high stability, pFGF1 is more potent than WFGF1.

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