Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent me... more Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as ZnII, CuI, and CdII that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the βE-domain of the early cysteine-labeled protein (Ec-1) from wheat (βE-Ec-1), which represents the first three-dimensional structure of a plant MT. The βE-domain comprises the 51 C-terminal residues of Ec-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear ZnII binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three ZnII ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys–metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15N relaxation experiments support the results of the structure determination in that βE-Ec-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced ZnII with CdII ions and examined the effects of metal ion release on incubation with a metal ion chelator.
Fusion protein purification systems based on self-cleavable protein splicing elements are well es... more Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.
Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn II an... more Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn II and Cu I ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn II-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn II competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the E c subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn II release processes. The Cu I-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn II release experiments with the individual metal-binding domains of E c-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent me... more Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn II , Cu I , and Cd II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β E-domain of the early cysteine-labeled protein (E c-1) from wheat (β E-E c-1), which represents the first three-dimensional structure of a plant MT. The β Edomain comprises the 51 C-terminal residues of E c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15 N relaxation experiments support the results of the structure determination in that β E-E c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn II with Cd II ions and examined the effects of metal ion release on incubation with a metal ion chelator.
JBIC Journal of Biological Inorganic Chemistry, 2007
Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary ... more Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (E c-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant E c-1 is able to bind a total of six divalent d 10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far. Keywords Metallothionein Á Metal thiolate cluster Á Zinc Á Cadmium Á Proteolytic digestion Abbreviations CD Circular dichroism DTT Dithiothreitol E c-1 Early cysteine-labeled protein ESI-MS Electrospray ionization mass spectrometry F-AAS Flame atomic absorption spectroscopy HEPES 4-(2-Hydroxyethyl)piperazine-1ethanesulfonic acid LMCT Ligand-to-metal charge transfer MCD Magnetic circular dichroism MT Metallothionein PAGE Polyacrylamide gel electrophoresis SDS Sodium dodecyl sulfate Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride Electronic supplementary material The online version of this article (
JBIC Journal of Biological Inorganic Chemistry, 2011
Metallothioneins (MTs) are small cysteine-rich proteins, coordinating various transition metal io... more Metallothioneins (MTs) are small cysteine-rich proteins, coordinating various transition metal ions including Zn II , Cd II , and Cu I. MTs are ubiquitously present in all phyla indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E c-1 from Triticum aestivum, common bread wheat, is a Zn II binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding E-domain was recently described. Here we present now also the structure of the N-terminal second domain, -E c-1, determined with NMR spectroscopy. The -E c-1 domain enfolds a M II 2 Cys 6 cluster and was characterized as part of the full-length Zn 6 E c-1 protein as well as in form of the separately expressed domain, both in the Zn II-and the Cd IIcontaining isoform. EXAFS analysis of Zn 2 -E c-1 clearly shows the presence of a ZnS 4 coordination sphere with average Zn-S distances of 2.33 Å. 113 Cd NMR experiments were used to identify the M II-Cys connectivity pattern, revealing two putative metal cluster conformations. In addition, the general metal ion coordination abilities of -E c-1 were probed with Cd II binding experiments as well as by pH titrations of the Zn II-and Cd II-forms, the latter suggesting an interaction of the and the E-domain within the full-length protein.
Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the h... more Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 wit...
JBIC Journal of Biological Inorganic Chemistry, 2007
The original publication was printed with a spelling error in the article title and in the abstra... more The original publication was printed with a spelling error in the article title and in the abstract part. In both instances it must read ''Triticum aestivum'' instead of ''Tricium aestivum''.
Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent me... more Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as ZnII, CuI, and CdII that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the βE-domain of the early cysteine-labeled protein (Ec-1) from wheat (βE-Ec-1), which represents the first three-dimensional structure of a plant MT. The βE-domain comprises the 51 C-terminal residues of Ec-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear ZnII binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three ZnII ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys–metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15N relaxation experiments support the results of the structure determination in that βE-Ec-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced ZnII with CdII ions and examined the effects of metal ion release on incubation with a metal ion chelator.
Fusion protein purification systems based on self-cleavable protein splicing elements are well es... more Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.
Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn II an... more Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn II and Cu I ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn II-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn II competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the E c subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn II release processes. The Cu I-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn II release experiments with the individual metal-binding domains of E c-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent me... more Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn II , Cu I , and Cd II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β E-domain of the early cysteine-labeled protein (E c-1) from wheat (β E-E c-1), which represents the first three-dimensional structure of a plant MT. The β Edomain comprises the 51 C-terminal residues of E c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15 N relaxation experiments support the results of the structure determination in that β E-E c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn II with Cd II ions and examined the effects of metal ion release on incubation with a metal ion chelator.
JBIC Journal of Biological Inorganic Chemistry, 2007
Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary ... more Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (E c-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant E c-1 is able to bind a total of six divalent d 10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far. Keywords Metallothionein Á Metal thiolate cluster Á Zinc Á Cadmium Á Proteolytic digestion Abbreviations CD Circular dichroism DTT Dithiothreitol E c-1 Early cysteine-labeled protein ESI-MS Electrospray ionization mass spectrometry F-AAS Flame atomic absorption spectroscopy HEPES 4-(2-Hydroxyethyl)piperazine-1ethanesulfonic acid LMCT Ligand-to-metal charge transfer MCD Magnetic circular dichroism MT Metallothionein PAGE Polyacrylamide gel electrophoresis SDS Sodium dodecyl sulfate Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride Electronic supplementary material The online version of this article (
JBIC Journal of Biological Inorganic Chemistry, 2011
Metallothioneins (MTs) are small cysteine-rich proteins, coordinating various transition metal io... more Metallothioneins (MTs) are small cysteine-rich proteins, coordinating various transition metal ions including Zn II , Cd II , and Cu I. MTs are ubiquitously present in all phyla indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E c-1 from Triticum aestivum, common bread wheat, is a Zn II binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding E-domain was recently described. Here we present now also the structure of the N-terminal second domain, -E c-1, determined with NMR spectroscopy. The -E c-1 domain enfolds a M II 2 Cys 6 cluster and was characterized as part of the full-length Zn 6 E c-1 protein as well as in form of the separately expressed domain, both in the Zn II-and the Cd IIcontaining isoform. EXAFS analysis of Zn 2 -E c-1 clearly shows the presence of a ZnS 4 coordination sphere with average Zn-S distances of 2.33 Å. 113 Cd NMR experiments were used to identify the M II-Cys connectivity pattern, revealing two putative metal cluster conformations. In addition, the general metal ion coordination abilities of -E c-1 were probed with Cd II binding experiments as well as by pH titrations of the Zn II-and Cd II-forms, the latter suggesting an interaction of the and the E-domain within the full-length protein.
Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the h... more Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 wit...
JBIC Journal of Biological Inorganic Chemistry, 2007
The original publication was printed with a spelling error in the article title and in the abstra... more The original publication was printed with a spelling error in the article title and in the abstract part. In both instances it must read ''Triticum aestivum'' instead of ''Tricium aestivum''.
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