Abstract
The plant metallothionein 2 protein from Cicer arietinum (cicMT2) is a typical member of the plant MT subfamily p2 that is characterized by an N- and C-terminal cysteine- (Cys-)rich, metal binding sequence connected by a long cysteine-free linker region. cicMT2 coordinates up to five ZnII or CdII ions by its 14 cysteine thiolate groups forming a single metal-thiolate cluster. While MTs from other phyla are considerably well-studied, many details about plant MTs are missing. In this study the metallation pathway of cicMT2 is investigated using mass spectrometry. To evaluate the influence of the linker region as well as the interplay of the two Cys-rich stretches, the full-length cicMT2 protein as well as the individual Cys-rich domains with and without the linker region were analysed. Up to three CdII ions can be coordinated by the eight Cys residues of the N-terminal part and up to two CdII ions by the six Cys residues of the C-terminal sequence. However, no preferential binding to either of the two sequences is observed, which is in-line with the closely similar apparent binding constants of the individual domains obtained from competition reactions with the chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid. The combination of limited proteolytic digestion, mass spectrometry, dynamic light scattering, size-exclusion chromatography, and 19F NMR spectroscopy enables us to draw conclusions about the overall protein-fold and the cluster formation process.
Salim, Alma