Date of Graduation

12-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Heyes, Colin D.

Committee Member

Thallapuranam, Suresh

Second Committee Member

Millett, Francis S.

Third Committee Member

Adams, Paul D.

Fourth Committee Member

Wang, Feng

Keywords

Fluorescence Spectroscopy; Fluorescent Dyes; FRET Assays; Human Fibroblast Growth Factor; Photophysics Properties; Single Molecule Spectroscopy

Abstract

Human fibroblast growth factor one (hFGF1) belongs to a family of 22 FGF members produced by fibroblast cells. Cell signaling during physiological processes of angiogenesis and wound healing occurs when hFGF1 binds to its receptor (FGFR). However, when heterogenous homeostasis is not maintained, fibroblast cells exhibit excessive proliferation which can lead to a myriad of cancers. smFRET is an ultrasensitive distant dependent (1-10 nm) technique capable of resolving such heterogeneity in structural dynamics and binding affinities (Kd). Therefore, we successfully designed and characterized fluorescently labeled hFGF1 tracers which span the visible light region of the electromagnetic spectrum for use in smFRET. Fluorescent labeling was achieved by utilizing the maleimide moiety of the dyes (Alexa Fluor, Cyanine and iFluor) to conjugate to the cysteine residues via a thioether bond. We synthesized biologically active, and site specifically fluorescently labeled hFGF1 mutants of F2C-hFGF1 (on the flexible N terminal loop) and T79C-hFGF1 (on the rigid loop between β strand 7-8). The main findings showed that the dye and protein required a certain degree of flexibility to obtain high labeling efficiency but having too much flexibility caused low labeling efficiency because of entropic penalty during conjugation. Ensemble and single molecule level characterization of the photophysical parameters of ensemble QY, fluorescence lifetime and molecular brightness revealed that the dyes properties depended on the rotational and vibrational degrees of freedom of the dye on the protein, dye structure and degree of flexibility of the labeling site. Overall, we successfully fluorescently labeled hFGF1 at specific sites and characterized its photophysical properties as this information is critical in designing FRET assay.

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