An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins

Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein specie

Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1

The early cysteine-labeled metallothionein (MT) from Triticum aestivum (common wheat), denoted Ec-1, features two structurally well-defined domains, γ and βE, coordinating two and four Zn(II) ions, respectively. While the protein is currently assumed to function mainly in zinc homeostasis, a low amount of copper ions was also recently detected in a native Ec-1 sample. To evaluate the observed copper binding in more detail, the recombinant Zn6Ec-1 form was exposed to different amounts of Cu(I) ions and the resulting species characterized with spectroscopic methods. Data reveal that the first Cu(I) equivalent coordinates exclusively to the N-terminal γ-domain of the protein and replaces one Zn(II) ion. To analyze the ability of the γ-domain for coordination of monovalent metal ions in more detail, the γ-Ec-1 peptide fragment was incubated with increasing amounts of Cu(I) and the process monitored with UV–VIS, circular dichroism, and luminescence spectroscopy. Closely similar spectra are observed regardless if the apo- or the metal ion-loaded and, hence, pre-folded forms, were used for the titration experiments with Cu(I). The results indicate that low amounts of Cu(I) ions displace the two metal ions subsequently and stoichiometrically, despite the different coordination geometry requirements of Cu(I) and Zn(II)

Investigating the influence of histidine residues on the metal ion binding ability of the wheat metallothionein γ-Ec-1 domain

While Zn(II) and Cd(II) have similar geochemical and environmental properties, their biological properties are distinctively different as Cd(II) ions have very limited metabolic significance and are mostly even toxic, while Zn(II) ions belong to the most essential micronutrients. One of the key proteins involved in intracellular Zn(II) and Cd(II) binding are metallothioneins (MTs), small cysteine-rich proteins ubiquitously found in many different organisms. In the past two decades, also MT sequences from diverse species that contain histidine residues have been found, and His-metal ion coordination has been shown. It is not clear, however, why in some MTs parts of the Cys residues are replaced by His, while most other MTs only contain Cys residues for metal ion binding. To address this question, we used the γ-domain of the early-cysteine labeled (Ec-1) metallothionein from common wheat as a model system because its enclosed M2Cys6 cluster represents the smallest metal-thiolate cluster possible with divalent metal ions. Based on the known three-dimensional structure of the γ-domain we set about to investigate the influence of a single Cys-to-His mutation on the structure and metal ion binding abilities of this domain. Combined data obtained by mass spectrometry, UV, as well as NMR spectroscopy suggest a preference for Zn(II) versus Cd(II) ions in the histidine containing binding site

An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins

Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-E$_c$-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species

Solution structure of the circular γ-domain analog from the wheat metallothionein Ec-1

The first cyclic analog of a metallothionein (MT) was prepared and analyzed by UV and (magnetic) circular dichroism spectroscopy, ESI-MS as well as NMR spectroscopy. Results reveal that the evaluated cyclic g-Ec-1 domain of the wheat MT Ec-1 retains its ability to coordinate two Zn(II) or Cd(II) ions and adopts a three-dimensional structure that is highly similar to the one of the linear wild-type form. However, the reduced flexibility of the protein backbone facilitates structure solution significantly and results in a certain stabilization of metal binding to the protein