Calcium (Ca2+), a crucial effector for many biological systems, has been associated with diseases such as cardiovascular disease, Alzheimer’s, Parkinson’s, cancer, and osteoporosis. It is important to develop calcium sensors to measure intracellular Ca2+ dynamics at various biological and pathological states. Our lab has engineered such probes by designing a Ca2+ binding site into fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) and mCherry. In this thesis, we aim to improve optical properties and metal binding properties of green EGFP-based sensor CatchER and mCherry based red sensors by site-directed mutagenesis and protein engineering, various spectroscopic methods and cell imaging. The green EGFP-based sensor CatchER, with a Ca2+ binding pocket charge of -5, displays the greatest affinity for Ca2+ and has the greatest fluorescence intensity change with Ca2+ when compared to its variants with a less negative binding pocket charge. In addition, we have also designed several SR/ER targeting CatchER variants using Ryanodine receptor and Calnexin transmembrane domains. These constructs were shown to display a strong presence in the SR/ER lumen and further designed for a new luminal orientation. Further, we have shown that the optical properties of two red calcium sensors can be significantly improved by modifying the local environment of the chromophore.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:scholarworks.gsu.edu:chemistry_theses-1092 |
| Date | 12 August 2016 |
| Creators | Goolsby, Demesheka |
| Publisher | ScholarWorks @ Georgia State University |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Chemistry Theses |
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