Digitized Honors Theses (2002-2017)

Date of Award

5-2016

Document Type

Undergraduate Thesis

Degree Name

BS

Department

Chemical Engineering

Faculty Mentor

Thomas Rich, Ph.D.

Advisor(s)

Silas Leavesley, Ph.D., Christy West, Ph.D.

Abstract

Forster Resonance Energy Transfer (FRET) is the process by which non-radiative energy transfer occurs between two molecules (the donor and the acceptor) with coupled dipole moments resulting in sensitized emission from the acceptor after donor stimulation. In the past 20 years, FRET has become a staple of molecular biology since FRET can be used to measure sub-optical distances and to spatiotemporally visualize cAMP signaling dynamics. The CFP­ EPAC-YFP protein is a FRET probe that can measure cAMP concentrations by reduction of FRET efficiency upon cAMP binding. Since cAMP signaling is implicated in cell differentiation, growth, and apoptosis, experimentalists continue to push the limits of the CFP­ EPAC-YFP probe to study cAMP dynamics under unusual physiological conditions. While CFP, EPAC, and YFP have been characterized separately, this study attempts to determine to the effect of pH, temperature, and reactive oxidative stress (ROS) variations on the FRET efficiency on the probe itself. Increasing temperatures led to increased FRET efficiency of the probe. This may be due to a reduction in viscosity and corresponding increase in diffusivity leading to increased thermal quenching of CFP and increased FRET to YFP. Titration of the probe around the pKa of YFP (7.0) caused the probe's FRET efficiency to exhibit a switch-like behavior around YFP's pKa due to the deprotonation of the fluorophore's histidine residue. Increasing hydrogen peroxide concentration to simulate ROS resulted in a decline in the overall intensity of both fluorophores due to oxidative bleaching, but YFP was more significantly affected resulting in a decrease in FRET efficiency.

Comments

© 2016 Robert Mines ALL RIGHTS RESERVED

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