Recombinant Rabbit Metallothionein-2C

What is Metallothionein-2 and how is its structure characterized?

Metallothioneins are proteins with high cysteine content that bind various heavy metals and are transcriptionally regulated by both heavy metals and glucocorticoids . The MT2 (also known as MT2A) isoform contains multiple cysteine residues that coordinate metal ions through thiolate bonds. The distinctive structural feature of metallothioneins is their ability to form metal-thiolate complexes where metal ions are coordinated by the sulfur atoms of cysteine residues .

The tertiary structure of metallothioneins typically consists of two domains (α and β) with metal-binding clusters. For rabbit MT-2, as with other mammalian MT-2 proteins, the conserved cysteine residues are critical for metal coordination, while the non-cysteine residues may influence metal binding specificity .

What metals does Rabbit MT-2 typically bind and with what stoichiometry?

Rabbit MT-2, like other mammalian metallothioneins, typically binds divalent metal ions such as Zn²⁺ and Cd²⁺, as well as monovalent Cu⁺. The binding stoichiometry varies depending on the metal:

The stoichiometry is typically determined through analytical techniques such as electrospray ionization mass spectrometry (ESI-MS) and spectroscopic methods .

What expression systems are most effective for producing Recombinant Rabbit MT-2?

Based on research with metallothioneins from various species, the most effective expression systems for recombinant MT production include:

Bacterial Expression (E. coli):

• Advantages: High yield, simplicity, cost-effectiveness

• Considerations: Requires optimization of culture conditions with specific metal supplementation

• Metal loading can be controlled by supplementing the culture media with specific metal ions (Cd²⁺, Zn²⁺, or Cu²⁺)

Yeast Expression:

• Advantages: Eukaryotic environment, post-translational modifications

• Applications: Particularly useful for functional studies through complementation in MT-knockout yeast strains

• Can validate metal specificity in a eukaryotic cellular environment

Experimental evidence indicates that metal-MT complexes synthesized in these heterologous hosts exhibit features equivalent to native complexes, supporting their use for structural and functional studies .

How can metal loading be optimized during recombinant expression?

Metal loading optimization during recombinant expression involves:

• Metal supplementation in growth media:

  • Add specific metal salts (ZnSO₄, CdCl₂, or CuSO₄) at defined concentrations

  • Timing of metal addition is crucial (typically at induction of protein expression)

• Expression conditions:

  • Lower temperatures (16-25°C) can improve proper folding and metal incorporation

  • Induction with lower IPTG concentrations (0.1-0.5 mM) may enhance metal loading

• Post-expression processing:

  • Metal exchange can be performed post-purification by:

    • Removing existing metals through acidification (pH 2-3)

    • Reconstituting with desired metals under controlled pH and aerobic/anaerobic conditions

The effectiveness of metal loading can be verified through mass spectrometry to confirm the formation of homometallic complexes with defined stoichiometry .

What spectroscopic techniques provide insight into metal binding by Rabbit MT-2?

Several spectroscopic techniques provide valuable information about metal-binding properties:

The spectral features of metal-MT complexes arise from the collective bonding of metals in oligonuclear metal thiolate complexes. For example, Cd₆-MT2 exhibits a steep rise in absorbance below 270 nm, typical of tetrahedral bonding of Cd²⁺ to multiple thiolate ligands, with associated intense positive and negative CD bands .

How can mass spectrometry be effectively applied to characterize Recombinant Rabbit MT-2?

Mass spectrometry offers powerful approaches for MT characterization:

• Native ESI-MS:

  • Preserves metal-protein complexes

  • Determines metal:protein stoichiometry

  • Identifies metallated species (e.g., Zn₁₋₇MT2)

• Denaturing MS conditions:

  • Assess protein integrity and modifications

  • Confirm primary sequence

• Bottom-up LC-MS approach:

  • Identification of specific metal-binding sites

  • Analysis of chemically labeled cysteine residues

  • Quantitative assessment of each cysteine's contribution to metal binding

• Combined MS and chemical labeling:

  • Using alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM)

  • Identifying accessible vs. metal-bound cysteines

  • Monitoring metal-binding mechanisms through changes in cysteine accessibility

An integrated approach combining these MS techniques can provide comprehensive insights into the metal-binding properties and mechanisms of Rabbit MT-2.

How does the mechanism of Zn(II) binding and unbinding to MT-2 occur?

The mechanism of Zn(II) binding and unbinding to MT-2 involves a coordinated process:

• Binding mechanism:

  • Sequential binding of Zn²⁺ ions to form Zn₁₋₆MT2 species

  • Cooperativity may exist between binding events

  • Formation of tetrahedral coordination geometry with four cysteine thiolates

• Unbinding mechanism:

  • pH-dependent release of Zn²⁺ ions

  • Competition with other metal ions (e.g., Cd²⁺ can displace Zn²⁺)

  • Potential redistribution of remaining metals among binding sites

• Experimental approach to study the mechanism:

  • Chemical labeling with iodoacetamide (IAM) to track free cysteine residues

  • Titration of apoMT2 with increasing Zn²⁺ equivalents

  • Monitoring changes in molar absorption coefficients through spectrophotometric titrations

  • Bottom-up proteomics analysis to determine the contribution of each cysteine residue to binding

Research indicates that Cys residues are labeled by IAM independently of protein conformational changes upon Zn²⁺ binding, and the binding mechanism can be followed by monitoring spectroscopic changes during metal titration .

What role do non-cysteine residues play in determining metal specificity?

While cysteine residues provide the direct coordination to metals, non-cysteine residues play crucial roles in determining metal specificity:

• Structural constraints:

  • Non-cysteine residues can constrain the spatial arrangement of cysteine residues

  • They may influence the preferred coordination geometry for different metals

• Evolutionary significance:

  • The complete sequential identity of cysteine residues across MT isoforms with different metal preferences suggests that non-cysteine residues are key determinants of specificity

  • Studies with pulmonate snail MTs demonstrate that evolutionary variation of non-cysteine residues can impose metal-specific character on coordination chemistry

• Proposed mechanism:

  • Due to their particular position in the sequence and chemical nature of their side chains

  • Non-cysteine amino acids constrain the sulfur ligands to assume only one of several theoretically possible spatial coordination foldings

  • This creates preferential binding environments for specific metals

This understanding suggests that strategic modification of non-cysteine residues could potentially engineer MT variants with altered metal specificity.

How can functional metal specificity be assessed through complementation studies?

Functional metal specificity can be rigorously assessed through yeast complementation studies:

• Experimental approach:

  • Transform yeast cells deficient in endogenous MTs (Cup1 and Crs5 knockout cells) with cDNAs coding for the MT of interest

  • Challenge transformed cells with increasing concentrations of different metals (e.g., Cu²⁺, Cd²⁺)

  • Compare growth rates to assess metal tolerance

  • Use appropriate controls (e.g., yeast Cup1, Crs5, mouse MT1)

• Interpretation of results:

  • Enhanced tolerance to specific metals indicates functional specificity

  • For example, snail HpCuMT conferred high copper tolerance but no cadmium tolerance

  • Conversely, snail HpCdMT provided cadmium detoxification capacity but little copper protection

• Advantages:

  • Assesses functional performance in a eukaryotic cellular environment

  • Validates in vitro binding preferences in a biological context

  • Provides insights into potential physiological roles

These complementation studies demonstrate that metal-specific binding preferences observed in recombinant systems translate to metal-specific functions in cellular environments.

What computational approaches complement experimental studies of Rabbit MT-2?

Computational approaches provide valuable insights when integrated with experimental data:

• Molecular Dynamics (MD) simulations:

  • Model building of fully metallated systems (e.g., Zn₇MT2)

  • Simulation of metal binding/unbinding events

  • Analysis of conformational changes upon metal binding

  • Force fields such as AMBER FF19SB with specific parameterization for metal ions and coordinating residues are suitable

• Density Functional Theory (DFT) calculations:

  • Quantum mechanical modeling of metal-thiolate clusters

  • Prediction of coordination geometries and energetics

  • Calculation of spectroscopic properties

• Integrated computational-experimental approaches:

  • Combining mass spectrometry data with MD simulations

  • Using spectroscopic data to validate computational models

  • Predicting effects of mutations on metal binding properties

An integrated approach combining these computational methods with experimental techniques provides a comprehensive understanding of the thermodynamic properties and structural dynamics of Zn₁₋₆MT2 species .

What can be learned from comparing Rabbit MT-2 with metallothioneins from other species?

Comparative analysis of metallothioneins across species provides evolutionary insights:

• Conservation patterns:

  • Cysteine residues are highly conserved across species

  • Non-cysteine residues show greater variability

  • These patterns suggest functional constraints on metal coordination sites

• Evolutionary mechanisms:

  • Gene duplication appears to be a common mechanism driving MT isoform differentiation

  • Following duplication, genes can independently accumulate mutations

  • Selection can then act on these variations to develop metal specificity

• Structure-function relationships:

  • Comparing MTs with different metal preferences (e.g., Cu-specific vs. Cd-specific)

  • Identifying sequence determinants of metal specificity

  • Understanding how evolutionary pressures shaped metal binding properties

This comparative approach provides a framework for understanding how metallothioneins evolved specialized functions while maintaining their fundamental metal-binding capabilities.

What is the significance of MT-2 pseudogenes in evolutionary studies?

MT-2 pseudogenes provide valuable insights into evolutionary processes:

• Features of MT-2 pseudogenes:

  • The rabbit metallothionein-2 pseudogene (MT-2ψ) shows evidence of complex rearrangements including recombination and deletion events

  • Lacks intervening sequences, 3' poly A tract, and 5' regulatory DNA sequences

  • Flanked by direct repeats that likely served as insertion sites

• Evolutionary implications:

  • MT-2ψ exhibits features of a processed retrogene

  • Provides evidence for retrotransposition events in MT evolution

  • Suggests mechanisms for gene family expansion and diversification

• Research applications:

  • Dating evolutionary events in MT gene family

  • Understanding selection pressures on functional vs. non-functional MT genes

  • Tracing the history of duplications, insertions, and rearrangements

Studying these pseudogenes alongside functional MT genes provides a more complete picture of the evolutionary history and diversification of the metallothionein gene family.