Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Abstract
:1. Introduction
2. Results and Discussion
2.1. Characterization of the Metallothionein (MT) System of Littorina littorea
2.2. The LlwtMT, Lltr1MT and Lltr2MT Recombinant Polypeptides
2.3. Zn(II) and Cd(II) Binding Capabilities of LlwtMT, Lltr1MT, and Lltr2MT
2.4. Cu(I) Binding Capabilities of LlwtMT, Lltr1MT, and Lltr2MT
3. Materials and Methods
3.1. Confirmation of the MT System of Littorina littorea
3.2. Construction and Cloning of the cDNAs Encoding the LlwtMT, Lltr1MT, and Lltr2MT Proteins
3.3. Synthesis and Purification of the Recombinant Zn-, Cd-, and Cu-Complexes of LlwtMT and the Lltr1MT and Lltr2MT Mutants
3.4. Zn(II)/Cd(II) and Zn(II)/Cu(I) Replacement Reactions in the Zn(II)-LlMT Proteins
3.5. Spectroscopic Analyses (ICP-AES, UV-Vis and CD) of the Metal Complexes Formed by the Llmt Proteins
3.6. Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI-TOF MS) of the Metal Complexes Obtained from the LlMT Proteins
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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MT | ICP-AES a | Neutral ESI-MS b | Experimental MM c | Theoretical MM d |
---|---|---|---|---|
LlwtMT | 9.5 | Zn9-MT | 10,753 | 10,754.1 |
Lltr1MT | 5.9 | Zn6-MT | 6807 | 6806.7 |
Lltr2MT | 6.1 | Zn6-MT | 7226 | 7227.2 |
LlwtMT | 8.7 | Cd9-MT | 11,177 | 11,177.3 |
Lltr1MT | 6.3 | Cd6-MT | 7089 | 7088.8 |
Lltr2MT | 6.5 | Cd6-MT | 7509 | 7509.3 |
MT | ICP-AES a | Neutral ESI-MS b | Exp MM c | Theor MM d | Acidic ESI-MS b | Exp MM c | Theor MM d |
---|---|---|---|---|---|---|---|
LlwtMT | 4.0 Zn 13.3 Cu | M14-MT M13-MT M12-MT M15-MT | 11,058 10,995 10,934 11,122 | 11,059.3 10,996.7 10,934.2 11,121.8 | Cu12-MT | 10,934 | 10,934.2 |
Cu8-MT | 10,682 | 10,684.0 | |||||
Cu11-MT | 10,869 | 10,871.6 | |||||
Cu10-MT | 10,808 | 10,809.1 | |||||
Cu13-MT | 10,995 | 10,996.7 | |||||
Cu9-MT | 10,747 | 10,746.5 | |||||
Cu14-MT | 11,061 | 11,059.3 | |||||
Lltr1MT | 3.5 Zn 5.8 Cu | M8-MT M9-MT M10-MT M7-MT M6-MT | 6925 6988 7050 6862 6796 | 6926.8 6989.3 7051.9 6864.2 6801.7 | Cu4-MT | 6675 | 6676.6 |
apo-MT | 6425 | 6426.4 | |||||
Cu8-MT | 6925 | 6926.8 | |||||
Cu5-MT | 6738 | 6739.1 | |||||
Cu6-MT | 6801 | 6801.7 | |||||
Cu7-MT | 6865 | 6864.2 | |||||
Lltr2MT | 3.2 Zn 6.5 Cu | M8-MT M9-MT M10-MT M7-MT M6-MT | 7344 7407 7471 7285 7224 | 7347.2 7409.8 7472.3 7284.7 7222.1 | Cu4-MT | 7095 | 7097.0 |
apo-MT | 6845 | 6846.8 | |||||
Cu8-MT | 7344 | 7347.2 | |||||
Cu5-MT | 7161 | 7159.6 | |||||
Cu6-MT | 7220 | 7222.1 | |||||
Cu7-MT | 7286 | 7284.7 |
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Palacios, Ò.; Jiménez-Martí, E.; Niederwanger, M.; Gil-Moreno, S.; Zerbe, O.; Atrian, S.; Dallinger, R.; Capdevila, M. Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character. Int. J. Mol. Sci. 2017, 18, 1452. https://doi.org/10.3390/ijms18071452
Palacios Ò, Jiménez-Martí E, Niederwanger M, Gil-Moreno S, Zerbe O, Atrian S, Dallinger R, Capdevila M. Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character. International Journal of Molecular Sciences. 2017; 18(7):1452. https://doi.org/10.3390/ijms18071452
Chicago/Turabian StylePalacios, Òscar, Elena Jiménez-Martí, Michael Niederwanger, Selene Gil-Moreno, Oliver Zerbe, Sílvia Atrian, Reinhard Dallinger, and Mercè Capdevila. 2017. "Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character" International Journal of Molecular Sciences 18, no. 7: 1452. https://doi.org/10.3390/ijms18071452