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Permanent link (DOI): https://doi.org/10.7939/R3SN01F56

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Theses and Dissertations

Tuning the properties of protein-based biosensors by chromophore replacement strategies. Open Access

Descriptions

Other title
Subject/Keyword
Fluorescence
Biosensor
Chromophore
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Assempour, Nazanin
Supervisor and department
Robert E. Campbell
Examining committee member and department
Christopher W. Cairo (Chemistry)
Rylan Lundgren (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2014-08-29T15:56:46Z
Graduation date
2014-11
Degree
Master of Science
Degree level
Master's
Abstract
Optical imaging of membrane potentials has become an immensely popular approach to study neuronal activity. Over the past decades, a number of voltage indicators have been introduced including the voltage sensitive fluorescent protein (VSFP) and microbial rhodopsins. However, most of these sensors suffer from weak signal-to-noise ratios, low sensitivity and incompatibility for deep tissue imaging. The second Chapter of this thesis describes our current efforts on improving microbial rhodopsinbased voltage indicators by replacing the retinal cofactor with a red shifted analogue. We synthesized the red-shifted retinal analogue (S4) with higher quantum yield and extinction coefficient compared to the natural retinal. Next, we reconstituted the ARCH and PROPS variants using the synthetic retinal and obtained the proteins with remarkably red-shifted absorbance and fluorescence spectra. This result demonstrates that chromophore replacement is promising strategy for modulating the spectral and voltage properties for more sensitive action potential detection. In the third Chapter, we describe our attempts to extend the fluorescence modulation strategy to “empty” fluorescent protein barrels, by replacing the endogenous chromophore with a synthetic version (HBDI). We expect that a chromophore replacement strategy could help provide new imaging tools with improved fluorescent properties. We performed some modifications on FP barrels with the aim of allowing the synthetic chromophore to insert into the empty barrel. Unfortunately, we could not obtain any improvements on fluorescent properties of FPs by this strategy.
Language
English
DOI
doi:10.7939/R3SN01F56
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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