Biomedical Sciences ETDs

Publication Date

Spring 4-17-2017

Abstract

Fluorogen activating proteins (FAPs) are genetically encoded tags made from single chain antibody fragments (scFv) designed to bind fluorogens with high specificity. Both the fluorogen and FAP can be modified to provide flexibility in properties such as affinity, membrane permeability, spectra, and quantum yield. The fluorogen Malachite Green (MG) has two excitation peaks, the maximum at 630 nm and a secondary peak at 450 nm. The emission spectra of blue-emitting fluorescence proteins, such as mCerulean (mCer), overlap with the MG secondary peak, generating a FRET pair with large Stokes shift emission. Using 405 nm excitation of mCer, we observe acceptor sensitized emission at wavelengths greater than 650 nm with no spectral crosstalk between the donor and acceptor channels. Additionally, donor only controls can be acquired for all cells as the acceptor is not present until after the addition of the fluorogen, providing intra-cellular control.

The FAP-FRET system has been characterized using proof of principle constructs: FAP-mCer-transmembrane (TM) as a positive FRET control and FAP-TM-mCer as a negative FRET control and expressed in HeLa cells. Multiple MG derivatives were compared and imaging parameters were optimized to determine the optimal FRET pair. Analysis was performed using code written in Matlab to mask the cell membrane and quantify FRET efficiencies, based on donor intensity before and after addition of fluorogen. Data from several fluorogen showed high energy transfer efficiency (~30%) with the FAP-mCer-TM construct compared to negligible FRET (~4%) for FAP-TM-mCer. Additional techniques were performed to support the FRET efficiency data, including spectral imaging and FLIM, which also reported FRET efficiency around 30% with the positive constructs and negligible FRET with the negative constructs. The FAP-FRET system is currently being used to study the kinetics of signaling proteins within the FcεRI pathway.

Keywords

Fluorogen activating protein (FAP), FRET, Fluorescent protein

Document Type

Thesis

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Masters

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Dr. Diane Lidke

Second Committee Member

Dr. Angela Wandinger-Ness

Third Committee Member

Dr. Heather Ward

Fourth Committee Member

Dr. Bill Shuttleworth

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