Zinc transfer from the embryo-specific metallothionein E-C from wheat : a case study

The direct observation of binding and release of spectroscopically silent metal ions such as Zn2+ and Ca2+ by proteins has been challenging before the advent of native electrospray ionisation mass spectrometry. This report highlights the powerful capability of ESI-MS to provide insight into metalloprotein speciation that is independent of any spectroscopic property. Using the zinc-binding plant metallothionein E-C from wheat as a study case, we show that ESI-MS is unique amongst other techniques in capturing intermediary metallospecies that evolve during the course of metal transfer to the chelator EDTA, as a model reaction to mimic the biological function of the protein as a zinc donor. Zinc release from the two-domain protein E-C appears to be extremely rapid and non-cooperative, and progresses with loss of one zinc ion from the fully loaded Zn-6 species, and a transient build-up of Zn-5 and Zn-4 species, which further react to give species with 0-3 zinc ions bound. H-1 NMR data has provided further insights into the different behaviour of the two domains upon metal depletion

Metallothioneins : unparalleled diversity in structures and functions for metal ion homeostasis and more

Metallothioneins have been the subject of intense study for five decades, and have greatly inspired the development of bio-analytical methodologies including multi-dimensional and multi-nuclear NMR. With further advancements in molecular biology, protein science, and instrumental techniques, recent years have seen a renaissance of research into metallothioneins. The current report focuses on in vitro studies of so-called class II metallothioneins from a variety of phyla, highlighting the diversity of metallothioneins in terms of structure, biological functions, and molecular functions such as metal ion specificity, thermodynamic stabilities, and kinetic reactivity. We are still far from being able to predict any of these properties, and further efforts will be required to generate the knowledge that will enable a better understanding of what governs the biological and chemical properties of these unusual and intriguing small proteins

Toward a property/function relationship for metallothioneins: Histidine coordination and unusual cluster composition in a zinc-metallothionein from plants

Early cysteine labeled (E-c) proteins are plant metallothioneins, which were first identified in wheat embryos and are thought to be seed-specific. An exhaustive analysis of expressed sequence tag (EST) entries reveals that homologs are expressed in embryos of both classes of flowering plants (monocotyledons and dicotyledons), but also occur in conifers (gymnosperms) and seed-free spike moss (lycophyta). Mass spectrometric and elemental analysis results indicate that, contrary to the widely propagated number of five, Ec binds predominantly six zinc ions in at least two zinc-thiolate clusters. H-1 and Cd-111 NMR experiments suggest that, in contrast to the majority of previously characterized metallothioneins, two conserved histidine residues participate in metal binding. The collected data is con- sistent with the presence of clusters unprecedented in metallothioneins so far. This novel cluster composition is accompanied by metalbinding properties that are substantially different from other metallothioneins; thus wheat EC binds zinc less strongly than either mammalian or cyanobacterial MTs. The unique biochemical properties of wheat Ec render it ideally suited for a role in zinc donation to nascent proteins during seed development, a role that has been suggested based on the fact that EC is induced by the plant hormone abscisic acid, but not by heavy metals. Our results provide a step further toward developing a property/function relationship for metallothioneins

The isolated Cys(2)His(2) site in EC metallothionein mediates metal-specific protein folding

The selectivity of proteins involved in metal ion homeostasis is an important part of the puzzle to understand how cells allocate the correct metal ions to the correct proteins. Due to their similar ligand-binding properties, and their frequent co-existence in soils, essential zinc and toxic cadmium are a particularly challenging couple. Thus, minimisation of competition of Cd2+ for Zn2+ sites is of crucial importance for organisms that are in direct contact with soil. Amongst these, plants have an especially critical role, due to their importance for nutrition and energy. We have studied an embryo-specific, zinc-binding metallothionein (EC) from wheat by nuclear magnetic resonance, electrospray mass spectrometry, site-directed mutagenesis, and molecular modelling. Wheat EC exploits differences in affinities of Cys(4) and Cys(2)His(2) sites for Cd2+ and Zn2+ to achieve metal-selective protein folding. We propose that this may constitute a novel mechanism to discriminate between essential Zn2+ and toxic Cd2+

Tools for metal ion sorting : in vitro evidence for partitioning of zinc and cadmium in C. elegans metallothionein isoforms

In vitro evidence for the isoform-specific partitioning of cadmium and zinc ions between the two C. elegans metallothioneins is presented. This observation is discussed in terms of isoform-specific affinities towards zinc and cadmium and the implications of our study on the in vivo roles of C. elegans metallothioneins

Differential reactivity of individual zinc ions in clusters from bacterial metallothioneins

The bacterial metallothionein SmtA binds four zinc ions with high affinity and specificity in a Zn4S9N2 cluster. We have explored the reactivity of these zinc ions towards the metal-chelator EDTA. Under pseudo-first-order conditions, initial break-down of zinc-thiolate bonds is rapid, followed by several slower phases. The reaction with stoichiometric amounts of EDTA is relatively slow and has been followed by H-1 NMR and mass spectrometry. Both methods reveal that partially metallated intermediates occur during the reaction. Three- and two-metal species are observed in only minor amounts, whereas the Zn-1 species is dominant during the mid stages of the reaction, before complete metal depletion occurs. These results suggest that the zinc finger site in SmtA is not only inert towards metal exchange, but also more resilient towards chelating agents. The greater inertness of this site may help to maintain the protein fold during metal depletion, and allow subsequent facile metal uptake. Conversely, it is likely that the protein fold is the major contributor to the observed persistence of Zn(1)SmtA in this reaction. Mass spectrometric studies with His-to-Cys mutants of SmtA reveal that the primary site for EDTA attack is the His49-containing zinc site C, and that His40 has a major influence on the reactivity of three sites

Site-specific N-terminus conjugation of poly(mPEG(1100)) methacrylates to salmon calcitonin : synthesis and preliminary biological evaluation

Recent advances in polymerization strategies have led to the development of novel polymer (poly) peptide biohybrid materials with potential application in the field of macromolecular therapeutics. In this current work, comb-shaped alpha-aldehyde poly(monomethoxy polyethylene glycol)methacrylates (p(mPEG)MA) with molecular masses in the 6.5-109 kDa range were prepared and conjugated, via reductive amination, to the Cys1 N-terminus of salmon calcitonin (sCT), a calcitropic hormone currently administered for the treatment of a number of hypercalcaemia-related diseases. The conjugation site was determined by tryptic digestion of the sCT-p(mPEG(1100)) MA biohybrids in conjunction with LC-MS MALDI-TOF spectrometry. Preliminary in vitro biological tests show that the polymer conjugation does not interfere with the biological activity of the sCT

C. elegans metallothioneins : response to and defence against ROS toxicity

The genome of the nematode Caenorhabditis elegans encodes for two multifunctional metal binding metallothioneins (MTs), CeMT-1 and CeMT-2. Here we applied qPCR to identify a transcriptional up-regulation following the exposure to free radical generators (ROS) paraquat or hydrogen peroxide, a trend that was confirmed with Pmtl::GFP expressing alleles. The deletion of the MT loci resulted in an elevation of in vivo levels of hydrogen peroxide and exposure to ROS caused a reduction in total egg production, growth and life span in wild type nematodes, effects that were particularly pronounced in the CeMT-2 and double knockout. Moreover, in vitro incubation of recombinant MTs with hydrogen peroxide demonstrated the presence of direct oxidation of the CeMTs, with zinc released from both isoforms and concomitant formation of intra-molecular disulfides. Finally, metabolic profiling (metabolomic) analysis of wild type and MT knockouts in the presence/absence of oxidative stressors, confirmed the overall trend described by the other experiments, and identified 2-aminoadipate as a potential novel small-molecule marker of oxidative stress. In summary, this study highlights that C. elegans metallothioneins scavenge and protect against reactive oxygen species and potentially against oxidative stress, with CeMT-2 being more effective than CeMT-1