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.
Original language | English (US) |
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Pages (from-to) | 717-728 |
Number of pages | 12 |
Journal | Journal of Biological Inorganic Chemistry |
Volume | 15 |
Issue number | 5 |
DOIs | |
State | Published - Jun 2010 |
Bibliographical note
Funding Information: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).
Keywords
- Di-iron-oxo proteins
- Four-helix bundle
- Metalloprotein models
- Spectroscopic characterization
- Thermodynamic stability