Metallothionein dimerization evidenced by QD-based Förster resonance energy transfer and capillary electrophoresis

Metallothionein dimerization evidenced by QD-based Förster resonance energy transfer and capillary electrophoresis

Článek WoS

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd2+ ions between molecules of metallothionein, the QDs and Cy3 were close enough, enabling a FRET signal. The FRET efficiency was calculated to be in the range of 11–77%. The formation of MT dimers in the presence of Cd2+ ions was confirmed by MALDI-MS analyses. Finally, the process of oligomerization resulting in FRET was monitored by CE, and oligomerization of MT was confirmed.

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd2+ ions between molecules of metallothionein, the QDs and Cy3 were close enough, enabling a FRET signal. The FRET efficiency was calculated to be in the range of 11–77%. The formation of MT dimers in the presence of Cd2+ ions was confirmed by MALDI-MS analyses. Finally, the process of oligomerization resulting in FRET was monitored by CE, and oligomerization of MT was confirmed.

FRET;Quantum dots;Oligomerization;Capillary electrophoresis

FRET;Quantum dots;Oligomerization;Capillary electrophoresis

PAVELICOVÁ, K.; POMPEIANO VANÍČKOVÁ, L.; HADDAD, Y.; NEJDL, L.; ZÍTKA, J.; KOČIOVÁ, S.; MRAVEC, F.; VACULOVIČ, T.; MACKA, M.; VACULOVIČOVÁ, M.; ADAM, V.

2021

15.02.2021

Elsevier

Nizozemsko

0141-8130

International Journal of Biological Macromolecules

170

1

Nizozemsko

53

60

8

https://www.sciencedirect.com/science/article/pii/S0141813020352466

http://hdl.handle.net/11012/208291

Pavelicova_2021