Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1
Abstract
:1. Introduction
2. Results and Discussion
2.1. Addition of One Equivalent of Cu(I) to Zn6Ec-1
2.2. Identification of the Cu(I) Binding Domain
2.3. Spectroscopic Studies of Cu(I) Binding to the Separate γ-Ec-1 Domain
2.4. Metal Ion to Protein Stoichiometries of γ-Ec-1 Species Formed during the Titrations with Cu(I) Ions
2.5. Biologically Relevant or an Artifact?
3. Materials and Methods
3.1. Chemicals and Solutions
3.2. Protein Expression and Purification
3.3. Proteinase K Cleavage
3.4. Titration of γ-Ec-1 with Cu(I)
3.5. CD Spectroscopy
3.6. Mass Spectrometry
3.7. Luminescence Spectroscopy
3.8. DLS Measurements
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
2-PDS | 2,2′-dithio-dipyridine | F-AAS | Flame atomic absorption spectroscopy |
CD | Circular dichroism | LMCT | Ligand-to-metal charge transfer |
DLS | Dynamic light scattering | MT | Metallothionein |
ESI-MS | Electrospray ionization mass spectrometry | SEC | Size exclusion chromatography |
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+1 Cu(I) | +2 Cu(I) | +3 Cu(I) | |
---|---|---|---|
ε262 nm,M−1·cm−1 | 11,521 | 26,208 | 25,226 |
number −S− | 2.6 | 6.0 | 5.8 |
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Tarasava, K.; Loebus, J.; Freisinger, E. Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1. Int. J. Mol. Sci. 2016, 17, 371. https://doi.org/10.3390/ijms17030371
Tarasava K, Loebus J, Freisinger E. Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1. International Journal of Molecular Sciences. 2016; 17(3):371. https://doi.org/10.3390/ijms17030371
Chicago/Turabian StyleTarasava, Katsiaryna, Jens Loebus, and Eva Freisinger. 2016. "Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1" International Journal of Molecular Sciences 17, no. 3: 371. https://doi.org/10.3390/ijms17030371