Metal-binding properties and structural characterization of a self-assembled coiled coil: Formation of a polynuclear Cd–thiolate cluster Daniil V. Zaytsev a , Vasily A. Morozov a , Jiufeng Fan a , Xianchun Zhu a , Madhumita Mukherjee a , Shuisong Ni b , Michael A. Kennedy b , Michael Y. Ogawa a, ⁎ a Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403, United States b Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States abstract article info Article history: Received 22 August 2012 Received in revised form 22 October 2012 Accepted 23 October 2012 Available online 29 October 2012 Keywords: Coiled coil Metallopeptide Polynuclear metal cluster Metal binding Cd(II) Self-assembly 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 ex- clusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spec- troscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH 5.4 and 8.5. The Cd 2+ -binding properties of the AQ-C16C19 peptide were studied by ultraviolet–visible spectroscopy (UV–vis), electrospray ionization mass spec- trometry (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 1.85 Å resolution, revealed an adamantane-like con- figuration 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 exog- enous 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 pH 5.4 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 po- tential use in developing synthetic metalloenzymes and multi-electron redox agents. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Metalloproteins play important catalytic, structural, and regulatory roles in living organisms which reflects the rich chemistry of protein- bound metal ions [1–3]. During the last several decades, the field of metalloprotein design has produced many examples of synthetic metallopeptide and metalloprotein complexes which can serve as both structural and functional mimics of native biological metallocenters. An area of particular interest has been the introduction of metal-binding sites into the hydrophobic interiors of α-helical bundles and coiled coils. Examples of single metal ions that can be incorporated into coiled coils and helical bundles include heme centers [4] and mononuclear com- plexes of Zn 2+ [5–8], Cu 2+ [6,9,10], Cu + [11], Ni 2+ [10], Cd 2+ [12–20], Hg 2+ [12,14,16,17,20–26], As 3+ [17,20,26–28], Pb 2+ [18,20,29], and Bi 3+ [18]. Given these notable successes, the introduction of multinuclear metal complexes into synthetic proteins presents another challenging target to pursue. Metal clusters are of special interest due to their impor- tance in a variety of catalytic and metalloregulatory processes and exam- ples of polynuclear sites being incorporated into synthetic multi-helix bundles include a photoactive copper(I) polynuclear center [30,31] cata- lytically relevant iron-based complexes including Fe 4 S 4 clusters [32–34] and non-heme diiron centers [35,36]. In this work, we report the metal binding properties of a coiled coil system based on the designed amphiphilic peptide called AQ-C16C19 which forms a stable three-stranded coiled coil in both the absence and presence of soft metal ions. A preliminary study of the binding of Cd 2+ ions to the AQ-C16C19 coiled coil system showed that metal loading monitored by UV spectroscopy is followed by a subsequent conforma- tional change of the peptide backbone as observed by CD spectroscopy [37]. Here, a more thorough study is reported of this metal–peptide system which is found to contain a polynuclear cadmium–thiolate center within a triple-stranded α-helical coiled coil whose peptide backbones are significantly distorted from the idealized structure. 2. Experimental section 2.1. General considerations Reagents were used as received without further purification. All solutions were made using >17.5 MΩ deionized water. Manipula- tions involving peptide solutions were performed under anaerobic Journal of Inorganic Biochemistry 119 (2013) 1–9 ⁎ Corresponding author. Tel.: +1 419 372 0433; fax: +1 419 372 8569. E-mail address: mogawa@bgsu.edu (M.Y. Ogawa). 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.10.010 Contents lists available at SciVerse ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio