Date of Award
2021
Document Type
Masters Thesis
Degree Name
M.S.
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
Yan Qin
Second Advisor
Michelle Knowles
Third Advisor
Daniel Linseman
Fourth Advisor
Dinah Loerke
Keywords
Endoplasmic reticulum, Fluorescent probe, Zinc
Abstract
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.
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Drew Maslar
Provenance
Received from ProQuest
File Format
application/pdf
Language
en
File Size
65 pgs
Recommended Citation
Maslar, Drew, "Development of Endoplasmic Reticulum Targeted Probes and Red Fluorescent Probes for Detecting Zinc" (2021). Electronic Theses and Dissertations. 1959.
https://digitalcommons.du.edu/etd/1959
Copyright date
2021
Discipline
Molecular biology, Cellular biology, Biology