title: Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high-resolution mass spectrometry.
abstract: Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design owing to their structural robustness and versatility. Moreover, their characterization provides important insights into protein folding in general. ZnF motifs usually possess an exceptional specificity and high affinity towards Zn(II) ion to drive folding. While the Zn(II) ion is canonically coordinated by two cysteine and two histidine residues, many other coordination spheres also exist in small ZnFs, all having four amino acid ligands. Here we used high-resolution mass spectrometry to study metal ion binding specificity and primary coordination sphere robustness of a designed zinc finger, named MM1. Based on the results, MM1 possesses high specificity for zinc with sub-micromolar binding affinity. Surprisingly, MM1 retains metal ion binding affinity even in the presence of selective alanine mutations of the primary zinc coordinating amino acid residues.
authors.affiliation: Department of Chemistry, University of Eastern Finland, Joensuu, FI-80101, Finland.
authors.forename: Mikko
authors.initials: M
authors.lastname: Laitaoja
authors.affiliation: Department of Chemistry, University of Eastern Finland, Joensuu, FI-80101, Finland.
authors.forename: Sari
authors.initials: S
authors.lastname: Isoniemi
authors.affiliation: BioMediTech, University of Tampere, Tampere, FI-33520, Finland.
authors.forename: Jarkko
authors.initials: J
authors.lastname: Valjakka
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Szeged, Szeged, H-6720, Hungary.
authors.forename: István M
authors.initials: IM
authors.lastname: Mándity
authors.affiliation: Department of Chemistry, University of Eastern Finland, Joensuu, FI-80101, Finland.
authors.forename: Janne
authors.initials: J
authors.lastname: Jänis
mesh_terms: D019943:Amino Acid Substitution; D013058:Mass Spectrometry; D008958:Models, Molecular; D020125:Mutation, Missense; D017510:Protein Folding; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: D015032:Zinc
keywords: mass spectrometry; metal ion binding; protein design; protein folding; zinc finger