Biochemical and biophysical characterization of mutant T. durum metallothionein

Official URL: http://192.168.1.20/record=b1378251 (Table of Contents)

Metallothionein (MT) family is characterized by low molecular weight cysteine rich proteins that bind d10 metals through thiolate bonds (Vasak et al. 2000). MTs are found in wide range of organisms and their classification was based on the phylogenetic relationships and patterns of distribution of Cys residues along the MT sequences (Kojima et al. 1999). In this study, the aim was to determine structural and metal binding properties of mutant Triticum durum metallothionein (G65C) and to analyze differences in metal binding capacity between the mutant and the native durum metallothionein (dMT). A mutation was introduced into one of the cys motifs (C-X-C) at DNA level to mimic a mammalian motif (C-X-C-C). The 65th glycine was mutated to a cysteine and the mutated gene was expressed in E. coli as Glutathione S-Transferase (GST) fusion protein (GSTG65C). G65C was cleaved from GST, purified and demetallated (apo- G65C) for biochemical and biophysical characterization. G65C mutant showed a higher level of oligomerization and polydispersity, but the cadmium content was 5 Cd++/protein which is similar to that of native dMT. Homogeneous solutions of apo-G65C were used for reconstitution studies. Apoprotein was reconstituted with cadmium and zinc and changes in structure were monitored by CD and absorbance measurements. Fully cadmium loaded protein was significantly different from holo-G65C purified directly from E.coli. During reconstitution major changes were observed at 230 and 250 nm. The strong absorbance increase observed at 230 nm indicates that significant conformational rearrangements take place in the hinge region - connecting metal binding domains – as well as within the cys-rich domains. The folding process in vitro takes place in a nonlinear fashion and is different from that of native dMT with the bridging thiolates forming later. The structural model developed from SAXS measurements show that both apo- and holo- G65C have asymmetric structures, the apoprotein being more elongated (maximum dimension: ~6 nm for holoand ~10 nm for apo-G65C). The models are consistent with two cluster structure. Results presented show that metal binding capacity is not dependent only on cys quantity, but also on cys motifs.