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Spectroscopic identification of different types of copper centers generated in synthetic four-helix bundle proteins
Zitatschlüssel ISI:000224964500039
Autor Schnepf, R and Haehnel, W and Wieghardt, K and Hildebrandt, P
Seiten 14389-14399
Jahr 2004
ISSN 0002-7863
DOI 10.1021/ja0484294
Adresse 1155 16TH ST, NW, WASHINGTON, DC 20036 USA
Journal J. Am. Chem. Soc
Jahrgang 126
Nummer 44
Monat NOV 10
Zusammenfassung Using a combined rational-combinatorial approach, stable copper binding sites were implemented in template-assembled synthetic four-helix bundle proteins constructed by three different helices with only 16 amino acid residues. These peptides include two histidines and one cysteine at positions appropriate for coordinating a copper ion. Sequence variations of the helices were made in the second coordination shell or even more remote from the copper binding site (i) to increase the overall stability of the metalloproteins and (ii) to fine-tune the structure and properties of the copper center. As a result, ca. 90\% of the 180 proteins that were synthesized were capable to bind copper with a substantially higher specificity than those obtained in the first design cycle (Schnepf, R.; Horth, P.; Bill, E.; Wieghardt, K.; Hildebrandt, P.; Haehnel, W. J. Am. Chem. Soc. 2001, 123, 2186-2195). Furthermore, the stabilities of the copper protein complexes were increased by up to 2 orders of magnitude and thus allowed a UV-vis absorption, resonance Raman, electron paramagnetic resonance, and (magnetic) circular dichroism spectroscopic identification and characterization of three different types of copper binding sites. It could be shown that particularly steric perturbations in the vicinity of the His(2)Cys ligand set control the formation of either a tetragonal (type II) or a tetrahedral (type I) copper binding site. With the introduction of two methionine residues above the histidine ligands, a mixed-valent dinuclear copper binding site was generated with spectroscopic properties that are very similar to those of Cu-A sites in natural proteins. The results of the present study demonstrate for the first time that structurally different metal binding sites can be formed and stabilized in four-helix bundle proteins.
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