Date of Award

May 2024

Degree Type

Thesis

Degree Name

Master of Science

Department

Physics

First Advisor

Valerica Raicu

Committee Members

Sarah Vigeland, Ionel Popa

Abstract

Fӧrster or Fluorescence Resonance Energy Transfer (FRET) is a biological phenomenon that occurs when energy is transferred non-radiatively from an excited donor molecule to an unexcited acceptor molecule when they are a certain distance from each other. One method of conducting FRET experiments is using FRET spectrometry which was previously introduced by the Raicu Lab. This method generates histograms of FRET efficiencies at pixel level called FRET spectrograms, that are fitted with models to determine the quaternary structure of protein oligomers as opposed to traditional FRET experiments which average over all FRET efficiencies. Currently, FRET spectrometry is implemented with spectrally resolved fluorescence microscopy to allow the computation of FRET efficiencies where the size of the oligomer is unknown.Another technique, Fluorescence Lifetime Imaging Microscopy (FLIM) has been successfully used to compute FRET efficiencies by fitting fluorescence decay curves with one or two exponential fits in order to determine the fluorescence lifetimes in the presence and absence of the donors and acceptors. However, using FLIM to study oligomers of arbitrary size is limited by the prerequisite knowledge of the distinct oligomeric configurations, as well as the mathematical limitation of extracting more than two lifetimes from a single decay. The tiFRET method was developed to address these limitations by numerically integrating the fluorescence decay curves at each pixel in order to determine the pixel level FRET efficiencies and generate FRET spectrograms. The tiFRET method was initially tested on cytoplasmic constructs of Cerulean, Venus, and Amber and compared to the traditional FLIM measurements using one and two lifetime fits. This method was then applied to a biological system that was known to oligomerize and compared to traditional FRET spectrometry experiments. G protein coupled receptors (GPCRs) are the largest family of membrane proteins. These receptors mediate most cellular responses to external stimuli, making them ideal candidates for drug research and development. In this study, the human muscarinic acetylcholine receptor M2 (M2R), one of five human muscarinic acetylcholine receptors, a class A- rhodopsin like GPCR was used. M2R is primarily responsible for slowing down the heart rate by controlling the rate of depolarization of the cell membrane by causing an outward flow of potassium ions. There are various studies that show the tendency of the muscarinic receptors to oligomerize, which was ideal for this study. Using two variants of the green fluorescent protein (GFP2, green fluorescent protein and YFP, yellow fluorescent protein) as fluorescent markers, two-photon fluorescence microscopy was used to collect temporally resolved fluorescence images and spectrally resolved fluorescence images of live cells. The tiFRET method was applied to the temporally resolved fluorescence images to calculate the pixel level distributions of apparent FRET efficiencies and obtain FRET spectrograms. The spectrally resolved fluorescence images were analyzed using the currently established method of performing FRET spectrometry to obtain FRET spectrograms. FRET metahistograms were generated and fit with a theoretical model in order to determine the oligomeric configuration of M2R. Both metahistograms were compared to determine the viability of the novel tiFRET method. In this study, the results of this comparison and future research are presented.

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