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Copper Environment in Artificial Metalloproteins Probed by Electron Paramagnetic Resonance Spectroscopy B

Flores, Marco, Olson, Tien L., Wang, Dong, Edwardraja, Selvakumar, Shinde, Sandip, Williams, JoAnn C., Ghirlanda, Giovanna, Allen, James P.
The Journal of physical chemistry 2015 v.119 no.43 pp. 13825-13833
amino acids, binding sites, circular dichroism spectroscopy, copper, divalent metals, electron paramagnetic resonance spectroscopy, ligands, metalloproteins, pH, peptides, spectral analysis
The design of binding sites for divalent metals in artificial proteins is a productive platform for examining the characteristics of metal–ligand interactions. In this report, we investigate the spectroscopic properties of small peptides and four-helix bundles that bind Cu(II). Three small peptides, consisting of 15 amino acid residues, were designed to have two arms, each containing a metal-binding site comprised of different combinations of imidazole and carboxylate side chains. Two four-helix bundles each had a binding site for a central dinuclear metal cofactor, with one design incorporating additional potential metal ligands at two identical sites. The small peptides displayed pH-dependent, metal-induced changes in the circular dichroism spectra, consistent with large changes in the secondary structure upon metal binding, while the spectra of the four-helix bundles showed a predominant α-helix content but only small structural changes upon metal binding. Electron paramagnetic resonance spectra were measured at X-band revealing classic Cu(II) axial patterns with hyperfine coupling peaks for the small peptides and four-helix bundles exhibiting a range of values that were related to the specific chemical natures of the ligands. The variety of electronic structures allow us to define the distinctive environment of each metal-binding site in these artificial systems, including the designed additional binding sites in one of the four-helix bundles.