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postgraduate thesis: NTA-based agents for intracellular labeling of metal binding and His-tagged proteins
Title | NTA-based agents for intracellular labeling of metal binding and His-tagged proteins |
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Authors | |
Advisors | Advisor(s):Sun, H |
Issue Date | 2017 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Yang, Y. [阳娅]. (2017). NTA-based agents for intracellular labeling of metal binding and His-tagged proteins. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Fluorescent imaging is an indispensable and ubiquitous technique for real-time monitoring intracellular events with high spatial resolution. Site-specific covalent labeling for metal binding proteins using small fluorescent probes is a powerful and attractive technique to study the protein localizations and functions under physiological conditions, which will improve our understanding on the metal-protein interaction and the metallodrug mechanism, considering about one third of the proteins being revealed as metalloproteins.
In this thesis, a green small fluorescent probe NTA-Fl is designed and synthesized to expand the spectral range with our previously reported blue probe NTA-AC (or TRACER) and red probe NTA-AB. They consist of three similar fundamental functional units: a reporting fluorophore (coumarin, fluorescein or BODIPY), a metal chelating moiety (NTA) and a photoreactive anchor (arylazide). They are assigned to detect, visualize and analyze potential metal-binding and His-tagged proteins inside living cells without interrupting their original states. Upon chelation with metal ions, the metal-leading probes with perfect membrane permeability target intracellular metal-binding and His-tagged proteins rapidly. The successful targeting results in remarkable fluorescence enhancement and irreversible covalent binding on the targeted proteins upon UV irradiation, which makes the subsequent protein identification and analysis possible.
Cu2+-NTA-Fl was applied in HeLa cells to examine its applicability first. It’s proved that Cu2+-NTA-Fl can enter the HeLa cells and light up the copper binding proteins within 10 minutes. The ability was then verified with Cr3+-NTA-Fl in Hep G2 cells using confocal imaging. Ni2+-TRACER was validated first with typical nickel binding proteins HspA in vitro and in overexpressed E. coli cells. Ni2+-TRACER was therefore used to track the Ni-proteomes in H. pylori cells. The blue fluorescence inside H. pylori cells indicated the effective labeling. The following protein separation, identification and analysis confirmed its ability in digging out metalloproteins inside living systems. Cu2+-TRACER was also applied to visualize and identify Cu2+-proteomes in HeLa cells. This methodology could be extended to visualize and identify other metalloproteomes in different types of systems. Mn+-NTA-Fl and Mn+-TRACER have made the blue and green imaging of same metal-binding proteins in cells successfully, enabling the observation and separation of different metal-binding proteins within the same cell simultaneously in the near future.
Ni2+-NTA-Fl was used for the rapid covalent binding of His-tagged and His-rich proteins in living cells with intense fluorescence turn-on. The Ni2+-NTA-Fl probe was validated with His-tagged protein His-XPA and His-rich protein SlyD in vitro and in overexpressed E. coli cells. It was proved to be membrane permeable with low toxicity. Afterwards, His-XPA was applied to demonstrate the applicability of targeting His-tagged proteins in mammalian cells. The targeted His-tagged and His-rich proteins would be covalently anchored to the probe and could be further confirmed by following SDS gels.
These probes will open new horizons on the research of the large library of metalloproteins (including His-tagged proteins) as well as cell biology of metals in cells and tissues. |
Degree | Doctor of Philosophy |
Subject | Fluorescent probes Metalloproteins - Analysis |
Dept/Program | Chemistry |
Persistent Identifier | http://hdl.handle.net/10722/250781 |
DC Field | Value | Language |
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dc.contributor.advisor | Sun, H | - |
dc.contributor.author | Yang, Ya | - |
dc.contributor.author | 阳娅 | - |
dc.date.accessioned | 2018-01-26T01:59:32Z | - |
dc.date.available | 2018-01-26T01:59:32Z | - |
dc.date.issued | 2017 | - |
dc.identifier.citation | Yang, Y. [阳娅]. (2017). NTA-based agents for intracellular labeling of metal binding and His-tagged proteins. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/250781 | - |
dc.description.abstract | Fluorescent imaging is an indispensable and ubiquitous technique for real-time monitoring intracellular events with high spatial resolution. Site-specific covalent labeling for metal binding proteins using small fluorescent probes is a powerful and attractive technique to study the protein localizations and functions under physiological conditions, which will improve our understanding on the metal-protein interaction and the metallodrug mechanism, considering about one third of the proteins being revealed as metalloproteins. In this thesis, a green small fluorescent probe NTA-Fl is designed and synthesized to expand the spectral range with our previously reported blue probe NTA-AC (or TRACER) and red probe NTA-AB. They consist of three similar fundamental functional units: a reporting fluorophore (coumarin, fluorescein or BODIPY), a metal chelating moiety (NTA) and a photoreactive anchor (arylazide). They are assigned to detect, visualize and analyze potential metal-binding and His-tagged proteins inside living cells without interrupting their original states. Upon chelation with metal ions, the metal-leading probes with perfect membrane permeability target intracellular metal-binding and His-tagged proteins rapidly. The successful targeting results in remarkable fluorescence enhancement and irreversible covalent binding on the targeted proteins upon UV irradiation, which makes the subsequent protein identification and analysis possible. Cu2+-NTA-Fl was applied in HeLa cells to examine its applicability first. It’s proved that Cu2+-NTA-Fl can enter the HeLa cells and light up the copper binding proteins within 10 minutes. The ability was then verified with Cr3+-NTA-Fl in Hep G2 cells using confocal imaging. Ni2+-TRACER was validated first with typical nickel binding proteins HspA in vitro and in overexpressed E. coli cells. Ni2+-TRACER was therefore used to track the Ni-proteomes in H. pylori cells. The blue fluorescence inside H. pylori cells indicated the effective labeling. The following protein separation, identification and analysis confirmed its ability in digging out metalloproteins inside living systems. Cu2+-TRACER was also applied to visualize and identify Cu2+-proteomes in HeLa cells. This methodology could be extended to visualize and identify other metalloproteomes in different types of systems. Mn+-NTA-Fl and Mn+-TRACER have made the blue and green imaging of same metal-binding proteins in cells successfully, enabling the observation and separation of different metal-binding proteins within the same cell simultaneously in the near future. Ni2+-NTA-Fl was used for the rapid covalent binding of His-tagged and His-rich proteins in living cells with intense fluorescence turn-on. The Ni2+-NTA-Fl probe was validated with His-tagged protein His-XPA and His-rich protein SlyD in vitro and in overexpressed E. coli cells. It was proved to be membrane permeable with low toxicity. Afterwards, His-XPA was applied to demonstrate the applicability of targeting His-tagged proteins in mammalian cells. The targeted His-tagged and His-rich proteins would be covalently anchored to the probe and could be further confirmed by following SDS gels. These probes will open new horizons on the research of the large library of metalloproteins (including His-tagged proteins) as well as cell biology of metals in cells and tissues. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Fluorescent probes | - |
dc.subject.lcsh | Metalloproteins - Analysis | - |
dc.title | NTA-based agents for intracellular labeling of metal binding and His-tagged proteins | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Chemistry | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991043979539903414 | - |
dc.date.hkucongregation | 2017 | - |
dc.identifier.mmsid | 991043979539903414 | - |