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protein engineering, fluorescent protein, biosensor

Alford, Spencer Caleb

Supervisor and department: Dr. Robert E. Campbell Chemistry

Examining committee member and department: Dr. M. Glover Biochemistry Dr. T. Lowary Chemistry Dr. J. Pelletier Chemistry ; external University of Montreal Dr. J. Harrison Chemistry Dr. J. Gibbs-Davis Chemistry

Department: Department of Chemistry

Specialization:

Date accepted: 2012-08-28T08:58:21Z

Graduation date: 2012-11

Degree: Doctor of Philosophy

Degree level: Doctoral

Abstract: Fluorescent proteins FPs are indispensible biochemical tools. The concerted efforts of protein engineers have produced FPs spanning the visible colour spectrum. This wide variety of FPs has greatly facilitated the development of FP-based biosensors. However, researchers rely on relatively few fundamental biosensor design templates. Förster resonance energy transfer and bimolecular complementation are the principal FP-based technologies for live cell imaging of physiological events, such as changes in small molecule concentration, enzymatic activities, and protein-protein interactions. Although widely used, these techniques are often restrictive due to poor signal-to-noise ratios and irreversible sensing, respectively. Furthermore, examples of these biosensor strategies incorporating red FPs are limited.In this thesis we describe our efforts to address this shortcoming in the area of FP-based biosensors. We developed a dimerization-dependent red FP ddRFP that serves as an alternative template for biosensor construction. The prototype ddRFP was engineered from a homodimeric variant of a Discosoma red FP. Through extensive directed evolution the homodimer was converted into a fluorogenic obligate heterodimer. The reversible changes in fluorescence intensity that result from association of the ddRFP monomeric constituents, or the irreversible decrease that accompanies dissociation of covalently linked partners following linker cleavage, provides a useful spectroscopic signal for biosensing applications. Specifically, we demonstrated that ddRFP is useful for detecting in vitro protein-protein interactions, as well as imaging changes in calcium ion concentration and activation of caspase-3 in live cells.We also report the expansion of the ddFP colour palette through the development of green ddGFP and yellow ddYFP ddFP variants. These variants have several improvements relative to the ddRFP prototype including increased in vitro contrast and brightness for ddGFP, and a reduced pKa for ddYFP. While their utility for some live cell imaging applications is restricted due to low dissociation constants, ddGFP proved to be a useful fluorescent label of intermembrane contact sites between the endoplasmic reticulum and the mitochondrial network.

Language: English

DOI: doi:10.7939-R33B5WH13

Rights: Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.





Author: Alford, Spencer Caleb

Source: https://era.library.ualberta.ca/


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University of Alberta Development of fluorogenic fluorescent protein heterodimers by Spencer C.
Alford A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry ©Spencer C.
Alford Fall 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the authors prior written permission. Abstract Fluorescent proteins (FPs) are indispensible biochemical tools.
The concerted efforts of protein engineers have produced FPs spanning the visible colour spectrum.
This wide variety of FPs has greatly facilitated the development of FP-based biosensors.
However, researchers rely on relatively few fundamental biosensor design templates.
Förster resonance energy transfer and bimolecular complementation are the principal FP-based technologies for live cell imaging of physiological events, such as changes in small molecule concentration, enzymatic activities, and protein-protein interactions.
Although widely used, these techniques are often restrictive due to poor signal-to-noise ratios and irreversible sensing, respectively.
Furthermore, examples of these biosensor strategies incorporating red FPs are limited. In this thesis we describe our efforts to address this shortcoming in the area of FP-based biosensors.
We developed a dimerization-dependent red FP (ddRFP)...





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