title: Metal-binding properties and structural characterization of a self-assembled coiled coil: formation of a polynuclear Cd-thiolate cluster.
abstract: This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH5.4 and 8.5. The Cd(2+)-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and (113)Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85Å resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S(3)O geometry. The metal cluster was found to form cooperatively at pH5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents.
authors.affiliation: Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403, United States.
authors.forename: Daniil V
authors.initials: DV
authors.lastname: Zaytsev
authors.forename: Vasily A
authors.initials: VA
authors.lastname: Morozov
authors.forename: Jiufeng
authors.initials: J
authors.lastname: Fan
authors.forename: Xianchun
authors.initials: X
authors.lastname: Zhu
authors.forename: Madhumita
authors.initials: M
authors.lastname: Mukherjee
authors.forename: Shuisong
authors.initials: S
authors.lastname: Ni
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kennedy
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Ogawa
mesh_terms: D020816:Amino Acid Motifs; D040761:Biomimetic Materials / Q000737:chemistry*; D002104:Cadmium / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D056831:Coordination Complexes / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D056831:Coordination Complexes; D008667:Metalloproteins; D010455:Peptides; D013438:Sulfhydryl Compounds; D002104:Cadmium; D018698:Glutamic Acid; D003545:Cysteine