Metal ion release from metallothioneins: proteolysis as an alternative to oxidation

Metallothioneins (MTs) are among others involved in the cellular regulation of essential ZnII and CuI 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 ZnII-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. ZnII 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 Ec 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 ZnII release processes. The CuI-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. ZnII release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences to the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs

Tris is a non-innocent buffer during intein-mediated protein cleavage

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

Comparacion de dos metodos de campo para la determinación de la conductividad hidráulica saturada en suelos del Sinu (Córdoba, Colombia).

El presente estudio fue realizado con el objetivo de evaluar el desempeño de la metodología del infiltrómetro de tensión de disco, comparado con el infiltrómetro de anillos para determinar conductividad hidráulica saturada (Ks) en suelos del Sinú. Se seleccionaron dos suelos: un Endoacuept arcilloso localizado en la Granja de la Universidad de Córdoba y un Haplustepts arenoso ubicado en el corregimiento de Jaraquiel (Montería). En cada localidad se realizaron pruebas de infiltración simultáneas con ambos métodos hasta alcanzar la infiltración estacionaria para luego calcular los correspondientes valores de Ks. Los valores de Ks obtenidos con infiltrómetro de anillos fueron significativamente mayores a los conseguidos con el infiltrómetro de tensión de disco, debido probablemente a las diferencias en la profundidad de instalación de los equipos y la relativa mayor área cubierta por el infiltrómetro de anillos. En el suelo arcilloso los valores de Ks medidos con el infiltrómetro de anillos estuvieron en el rango de 1.29 a 6.02 cm h-1 y los medidos con el infiltrómetro de tensión de disco entre 0.74 y 2.64 cm h-1. En el suelo arenoso fueron de 4.25 a 24.15 cm h-1 con el infiltrómetro de anillos y de 2.23 a 7.87 cm h-1 con infiltrómetro de tensión de disco. El coeficiente de variación con el infiltrómetro de anillos fue del 47% y 52% en el suelo arcilloso y arenoso, respectivamente; mientras que con el infiltrómetro de tensión de disco se mantuvo en el 37% en los dos suelos. El método infiltrómetro de tensión de disco resulta ser más práctico por su facilidad, el tiempo, el número de operarios, el consumo de agua, el costo y menor variabilidad

The two distinctive metal ion binding domains of the wheat metallothionein Ec-1

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 with an excess of Zn(II) shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E(c)-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding

Protein and metal cluster structure of the wheat metallothionein domain gamma-Ec-1: the second part of the puzzle

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including ZnII, CdII, and CuI. MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT Ec-1 from Triticum aestivum, common bread wheat, is a ZnII- binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding beta-E domain was recently described. Here we present the structure of the N-terminal second domain, gama-Ec-1, determined by NMR spectroscopy. The gamma-Ec-1 domain enfolds an MIICys cluster and was characterized as part of the full-length Zn6Ec-1 protein as well as in the form of the separately expressed domain, both in the ZnII-containing isoform and the CdII-containing isoform. Extended X-ray absorption fine structure analysis of Zn2g- Ec-1 clearly shows the presence of a ZnS4 coordination sphere with average Zn–S distances of 2.33 A ̊. 113Cd NMR experiments were used to identify the MII-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of g-Ec-1 were probed with CdII binding experiments as well as by pH titrations of the ZnII and CdII forms, the latter suggesting an interaction of the c domain and the bE domain within the full-length protein

Structural features specific to plant metallothioneins

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn(II), Cd(II), and Cu(I). The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E(c)-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal-thiolate cluster structures