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

• Author / Creator

  Assempour, Nazanin

• 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.

• Subjects / Keywords

  • Fluorescence
  • Biosensors
  • Chromophore

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3SN01F56

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. 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.