Date of Award

May 2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physics

First Advisor

Valerică Raicu

Committee Members

David Frick, Ionel Popa, Marius Schmidt, Peter Schwander

Keywords

Fluorescent proteins, Förster Resonance Energy Transfer (FRET), FRET simulation, Photophysical effects of FPs, Protein-Protein Interactions, Two-photon micro-spectroscopy

Abstract

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photoswitching, is critical to the development of criteria for their selection as tags for fluorescence-based biological applications. My overall goal has been to study the photophysical properties of FPs under various excitation conditions, quantify the contributions that photophysical effects make to Förster resonance energy transfer (FRET) measurements, and provide appropriate experimental guidelines for FRET studies.Over the past two decades, we have witnessed a mounting interest in the study of protein localization and interactions using two-photon excitation (TPE) of fluorescently labeled proteins. While there is a plethora of information available regarding the photophysical and photochemical properties of commonly used fluorescent proteins when subjected to single-photon excitation, unfortunately, there is still very limited information regarding these properties under TPE. Therefore, I started my research by investigating the photophysical properties of several widely used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. The results provided in the first part of this dissertation indicate that a number of properties, including the excitation and emission spectra, the relative brightness, and the extent of photobleaching and photoswitching, are markedly different under TPE when compared to single-photon excitation. The second part of this dissertation describes a detailed study of the nature and degree of the contributions to FRET of the photophysical effects exhibited by FPs used as fluorescent tags for the proteins of interest. FRET is a widely used technique to study the quaternary structure of protein complexes (i.e., intermolecular distances and binding interfaces) in living cells. Critical to the interpretation of the results of such studies is the theoretical treatment of oligomers comprised of more than one donor and one acceptor that may exchange electronic excitations via FRET. The question has been theoretically addressed by developing the kinetic theory of FRET. However, there is no detailed analysis how FRET and the kinetic theory of FRET respond to photophysical effects such as photobleaching of the donor and acceptor tags. Herein, we have presented a comparative analysis of different protocols for calculation of the FRET efficiency. We studied the effects of changing the laser excitation power on FRET measurements by quantifying the deviations from the kinetic theory of FRET, which does not include photobleaching currently. We also used a simple but effective numerical method to estimate the degree to which photobleaching of donors and acceptors was responsible for the observed discrepancies between the two sets of FRET efficiencies. We found that under low excitation power, and with carefully selected excitation wavelengths, the FRET efficiency of an obligate trimeric construct, made by fusing one FRET donor and two FRET acceptors to one another, is in agreement within less than 2% with the FRET efficiency predicted by the kinetic theory of FRET. However, at higher excitation powers, the FRET efficiencies changed significantly, due to the photobleaching of both the donor, through direct excitation, and the acceptor, mostly through FRET-induced excitation. If ignored, these effects could cause systematic and random errors as large as 15% or more in the FRET efficiency values obtained from experiments, which would cause significant uncertainties regarding the quaternary structure to be determined. This study therefore provides critical information for selecting appropriate fluorescent proteins and experimental conditions for reliable FRET measurements in oligomeric complexes of associating molecules in living cells.

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