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Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

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Abstract

The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described (“DF” represents due ferri, Italian for “two iron,” “di-iron”). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop–helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV–vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di-Co(II)- and di-Fe(III)-DF proteins, including the presence of a μ-oxo-di-Fe(III) unit. Interestingly, UV–vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.

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Abbreviations

CD:

Circular dichroism

DF:

Due ferri

EPR:

Electron paramagnetic resonance

GdnHCl:

Guanidine hydrochloride

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

LMCT:

Ligand-to-metal charge transfer

PDB:

Protein Data Bank

RP-HPLC:

Reverse-phase high-performance liquid chromatography

TFA:

Trifluoroacetic acid

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Acknowledgments

The authors wish to thank Marco Trifuoggi for metal content analysis. This work was supported by the European Commission (Marie Curie Fellowship HPMD-GH-01-00113-03 to R.T.M.R.), the US National Institutes of Health (Grant GM-38767 to L.Q. and graduate traineeship GM-08700 to E.F.), and the Italian MIUR (PRIN 2007, prot. 2007KAWXCL).

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Author notes

  1. Rafael Torres Martin de Rosales

    Present address: Division of Imaging Sciences, King’s College London, St. Thomas’ Hospital, 4th Floor, Lambeth Wing, London, SE1 7EH, UK

Authors and Affiliations

  1. Department of Chemistry, University of Naples “Federico II”, Via Cintia, 80126, Naples, Italy

    Rafael Torres Martin de Rosales, Marina Faiella, Concetta Andreozzi, Vincenzo Pavone, Ornella Maglio, Flavia Nastri & Angela Lombardi

  2. Department of Chemistry, Centre for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN, 55455, USA

    Erik Farquhar & Lawrence Que Jr

  3. IBB, CNR, Via Mezzocannone 16, 80134, Naples, Italy

    Ornella Maglio

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  1. Rafael Torres Martin de Rosales
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Correspondence to Angela Lombardi.

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Torres Martin de Rosales, R., Faiella, M., Farquhar, E. et al. Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions. J Biol Inorg Chem 15, 717–728 (2010). https://doi.org/10.1007/s00775-010-0639-9

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