Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium stores, and gated by membrane calcium channels/pumps. To closely monitor calcium dynamics there is a pressing need to develop calcium sensors that are targeted to high calcium environment such as the ER/SR with relatively low binding affinity and fast kinetic properties to complement the current calcium indicator toolkits. In this dissertation, the development of fast red florescent calcium binding protein using the protein design is reported. The results show the calcium dependent fluorescence increase of mCherry mutant MCD1 (RapidER) and MCD15 (RapidER’) is able to monitor the ER calcium release in several cell lines responding to perturbations of extracellular calcium signaling. The specific targeting to the ER membrane was achieved by fusing the ryanodine receptor 1 transmembrane domains for the spatio-temporal calcium imaging.
To understand the underlying mechanism of calcium binding induced fluorescence increase in the designed calcium sensor CatchER, the fluorescence lifetime of CatchER was determined in calcium free and bound forms using time resolved florescence spectroscopy. The results suggest that calcium binding inhibits the geminate quenching, resulting in a longer lifetime when the anionic form is indirectly excited at 395 nm. It is believed that such unique calcium-induced lifetime change can be applied to monitor calcium signaling in cell imaging.
NMR spectroscopy was used to investigate the protein-protein/ligand interaction in this dissertation. The residual dipolar coupling and T1, T2, NOE dynamic study were carried out to understand the binding mode of CaM and the N-terminal intracellular loop of connexin 43. The results show that both N and C terminal domains of Ca2+-CaM contact with the peptide, leading to a partially unwound and bending central helix of CaM. The ligand binding induced conformational change was demonstrated by selectively labeled proteins including extracellular domain of calcium sensing receptor and the bacterial membrane protein SecA fragments C34 and N68.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:scholarworks.gsu.edu:chemistry_diss-1085 |
| Date | 18 December 2013 |
| Creators | Zhuo, You |
| Publisher | ScholarWorks @ Georgia State University |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Chemistry Dissertations |
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