Date of Award
Endoplasmic reticulum, Fluorescent probe, Zinc
Zinc (Zn2+) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn2+ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn2+, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn2+ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design to target the currently developed cpFP Zn2+ sensors to the lumen of the endoplasmic reticulum (ER), and expand the tool kit of cpFP Zn2+ sensors by introducing the first generation of red-shifted cpFP Zn2+ sensors. We demonstrate that not only can these Zn2+ sensors be targeted to the ER, but they can functionally be used to estimate labile ER Zn2+ concentration. We also show that red-shifted cpFP Zn2+ sensors display high sensitivity for detecting labile Zn2+, similar to the green-shifted cpFP Zn2+ sensors. These discoveries add to the current knowledge of labile Zn2+ within the lumen of the ER and introduce a new sensor that allows for the observation of labile Zn2+ in cells that was previously unavailable.
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Maslar, Drew, "Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc" (2021). Electronic Theses and Dissertations. 1959.
Received from ProQuest
Molecular biology, Cellular biology, Biology