One third of proteins in nature have been revealed as metalloproteins, whereas most of them remain uncharacterized, probably due to the lack of robust methods especially for tracking metalloproteins within the living context. Fluorescent labeling is capable to detect biomolecules with molecular resolution in living cells. Tracking metal-binding proteins in living cells by fluorescence could provide invaluable information in understanding their localization and potential functions in the native environment.
A synthetic molecular probe NTA-AC was designed and synthesized to track metal-associated proteins in living cells upon chelation with metal ions. The fluorescent probe consists of a small molecular fluorophores, a metal-chelating moiety to direct the metal-chelated probe to the protein targets, and a photo-active crosslinker. Metal being chelated could help further explore potential binding targets and direct the fluorescent agent to the appropriate region, then subsequently covalent linkage to targets could be generated through photo-activation. NTA-AC was therefore chelated with different metals to examine its binding preference to different proteins.
The Ni2+-chelating probe was applied to track Ni2+-binding proteins as an example to validate its applicability. Ni2+-NTA-AC preferentially binds to histidine-rich peptides and proteins thus verified its binding specificity. The Ni2+-chelated probe was further exploited to light up over-expressed histidine-rich proteins in Escherichia coli cells to validate its membrane permeability and binding specificity. In addition, the probe was applied to label His-tagged proteins expressed in tobacco plant cells to further evaluate its applicability in detecting and localizing the protein targets in eukaryotic cells. Afterwards, Ni2+-NTA-AC was exploited to track Ni2+-binding proteins in living Helicobacter pylori cells and incorporated with gel electrophoresis and mass spectrometry for protein identification. Many proteins identified are correlated to Ni2+-association and thus validating the applicability of the probe.
Bi3+-chelated NTA-AC was therefore used to mine potential targets in H. pylori. Intense fluorescence was observed within H. pylori cells thus indicating the effectiveness of the fluorescent labeling. Protein separation and identification was therefore initiated to trace potential targets, while finding that some of the Bi
3+-coordinated proteins participate in various functioning pathways of the pathogens. The effects of colloidal bismuth subcitrate (CBS) on pH buffering and redox defense systems were therefore determined and verified, confirming that respective proteins could be potential therapeutic targets of the drug.
Cr3+-NTA-AC was further applied to human Hep G2 cell line to determine Cr3+-binding targets in mammalian cells. Their localization on mitochondria was revealed, implying the potential effects of Cr3+ on mitochondria. Further confirmation of protein targets was performed through protein separation and identification. Proteins identified could be positively correlated to mitochondrial functions and thus revealing that Cr3+ might exert its effect at mitochondria. Addition of Cr3+ to Hep G2 could prevent mitochondrial fragmentation induced by hyperglycemia, which thus suggests the possible therapeutic function of Cr3+.
The extensive application of NTA-AC in tracking Ni2+-, Bi3+- and Cr3+-associated proteins has validated the effectiveness of such strategy in detecting and localizing metalloproteins within the living context and thus could be extended to investigate other metalloproteomes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/206989 |
| Date | January 2013 |
| Creators | Lai, Yau-tsz, 黎佑芷 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | Creative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| Relation | HKU Theses Online (HKUTO) |
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