Gordon W. Irvine

Cysteine accessibility during As3+ metalation of the α- and β-domains of recombinant human MT1a.

Metallothionein is a ubiquitous metal binding protein that plays an important role in metal ion homeostasis and redox chemistry within cells. Mammalian metallothioneins bind a wide variety of metals including the metalloid As3+ in two domains (β and α) connected by a short linker sequence. Three As3+ bind in each domain for a total of 6 As3+ per protein. In recombinant human metallothionein (rh-MT1a) each As3+ binds three cysteine residues to form As3Cys9(CysSH)2-α-rhMT1a in the 11 Cys α-domain and As3Cys9-β-rhMT1a in the 9 Cys β-domain. This means that there should be 2 free cysteines in the α-domain but no free cysteines in the β-domain. By using benzoquinone, the number and relative accessibility of the free cysteinyl thiols during the metalation reactions were determined. The electrospray ionization mass spectrometry (ESI-MS) data confirmed that each As3+ binds using exactly 3 cysteine thiols and showed that there was a significant difference in the reactivity of the free cysteines during the metalation reaction. After a reaction with two molar equivalents of As3+ to form As2Cys6(CysSH)3-αβ-rhMT1a, the remaining 3 Cys in the 9 Cys β-domain were far less reactive than those in the α-domain. Molecular dynamics calculations for the metalation reactions with As3+ measured by ESI-MS allowed an interpretation of the mass spectral data in terms of the relative location of the cysteine thiols that were not involved in As3+ coordination. Together, these data provide insight into the selection of a specific cysteinyl thiol by the incoming metals during the stepwise metalation of metallothioneins.

Metalation Kinetics of the Human α-Metallothionein 1a Fragment Is Dependent on the Fluxional Structure of the apo-Protein

Mammalian metallothioneins are cysteine rich metal-binding proteins comprising, when fully metalated, two metal-binding domains: the α-domain binds with M4(II)S(Cys)11 stoichiometry and the β domain binds as M3(II)S(Cys)9 stoichiometry. While the fully metalated species have been widely studied, the metalation of the apoprotein is poorly understood. Key to a description of the metalation pathway(s) is the initial conformation of the apoprotein and the arrangement of the metal-coordinating cysteines prior to metalation. We report the effect of the ill-defined, globular structure of apoMT on metalation rates. In order to overcome the experimental limitations inherent in structural determinations of a fluxional protein we used a detailed analysis of the apo-α-metallothionein conformation based on the differential rate of cysteine modification with benzoquinone. The ESI mass spectral data show the presence of two distinct conformational families: one a folded conformational family at neutral pH and a second an unfolded family of conformations at low pH. The Cd(II) metalation properties of these two conformationally distinct families were studied using stopped-flow kinetics. Surprisingly, the unfolded apoprotein metalated significantly slower than the folded apoprotein, a result interpreted as being due to the longer time required to fold into the cluster structure when the fourth Cd(II) binds. These results provide the first evidence for the role of the structure of the apo-αMT in the metalation reaction pathways and show that cysteine modification coupled with ESI-MS can be used to probe structure in cysteine-rich proteins.

Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange.

Mammalian metallothioneins (MTs) are small, metal binding proteins implicated in cellular metal ion homeostasis and heavy metal detoxification. Divalent, metal-saturated MTs form two distinct domains; the N-terminal β domain binds three metals using nine Cys residues, and the C-terminal α domain binds four metals with 11 Cys residues. Domain selection during zinc binding and cadmium exchange to human MT1A was examined using a series of competition reactions with mixtures of the isolated domain fragments. These experiments were conducted at two biologically significant pH conditions where MTs exist in vivo. Neither zinc binding nor cadmium exchange showed any significant degree of specificity or selectivity based on detailed analysis of electrospray ionization mass spectrometric and circular dichroic data. Under acidic conditions, zinc binding and cadmium exchange showed slight α domain selectivity because of the increased preference for cooperative clustering of the α domain. Modeling of the reactions showed that at both physiological (7.4) and acidic (5.8) pHs, zinc binding and cadmium exchanges occur essentially randomly between the two fragments. The metal binding affinity distributions between the domain fragments are comingled and not significantly separated as required for a domain specific mechanism. The models show rather that the order of the binding events follows the order of the binding affinities that are distributed across both domains and that this can be considered quantitatively by the KF(Cd)/KF(Zn) binding constant ratio for each metal bound.

Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites.

A number of biological functions have been ascribed to mammalian metallothioneins (MTs) including zinc and copper homeostatic regulation, redox activity and detoxification of heavy metals like cadmium and mercury. It is unclear how these small, fluxional, cysteine rich proteins manage to play each of these vital roles. Using a combination of cadmium and pH titrations of the isolated domains of human MT isoform 1a monitored by electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report the pH dependencies that control metal binding mechanisms of these domains. We report that the α-domain mechanism is driven by the cooperative formation of the Cd4MT cluster at slightly acidic pH (≤6.9) switching binding mechanisms over a physiologically relevant pH range, whereas the β-domain metalation mechanism is dominated by terminal coordination of cadmium in a non-cooperative manner above pH5.5. These results suggest that, in some acidic sub-cellular compartments, cadmium could be sequestered in the α-domain, leaving zinc or copper bound in the β-domain and available for donation to other metalloproteins. We propose that these results can be explained by the intrinsic nature of the two domains, the four-metal α-cluster being more resistant to proton attack due to its lower charge-to-metal ratio, compared with the three-metal β-domain.

Selective cysteine modification of metal-free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI-MS

Human metallothionein 1a, a protein with two cysteine‐rich metal‐binding domains (α with 11 Cys and β with 9), was analyzed in its metal‐free form by selective, covalent Cys modification coupled with ESI‐MS. The modification profiles of the isolated β‐ and α‐fragments reacted with p‐benzoquinone (Bq), N‐ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI‐mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.

A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury

Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor.

Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS), and step-wise “snapshot” ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation.

Chromatographic separation of similar post-translationally modified metallothioneins reveals the changing conformations of apo-MT upon cysteine alkylation by high resolution LC-ESI-MS

Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated β-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the β-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release.