Nickel(ii)-promoted specific hydrolysis of zinc finger proteins

Metallomics : Integrated Biometal Science
Agnieszka Belczyk-CiesielskaWojciech Bal

Abstract

In this work we demonstrate that the previously described reaction of sequence specific Ni(ii)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(ii) ions. It results in loss of the Zn(ii) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(ii)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(ii)-susceptible sequence between the edited zinc finger and an affinity tag allows for removal of the latter sequence by Ni(ii) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(ii) ions. Mass spectra revealed that a Ni(ii) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(ii)-dependent protein hydrolysis is influenced by the Ni(ii) concentration,...Continue Reading

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Citations

Oct 12, 2018·Metallomics : Integrated Biometal Science·C Abbehausen
May 13, 2020·Metallomics : Integrated Biometal Science·Nina Ewa WezynfeldWojciech Bal
Jan 19, 2022·Angewandte Chemie·Katarzyna KluskaArtur Krężel

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Methods Mentioned

BETA
affinity purification
electrophoretic methods
circular dichroism
electrophoretic mobility shift
electrophoresis
Circular
Electrophoretic mobility shift assay

Software Mentioned

Zinc Finger Tools

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