Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions.
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 mu-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.
Department of Chemistry, University of Naples "Federico II", Italy.
Rafael
R
Torres Martin de Rosales
Marina
M
Faiella
Erik
E
Farquhar
Lawrence
L
Que
Concetta
C
Andreozzi
Vincenzo
V
Pavone
Ornella
O
Maglio
Flavia
F
Nastri
Angela
A
Lombardi
D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D055550:Protein Stability; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D001386:Azides; D008667:Metalloproteins; D008670:Metals; D003035:Cobalt; D008345:Manganese; D007501:Iron; D015032:Zinc