| 20060593 |
Xie F, Sutherland DE, Stillman MJ, Ogawa MY: Cu (I) binding properties of a designed metalloprotein. J Inorg Biochem. 2010 Mar;104(3):261-7. Epub 2009 Dec 16.
The Cu (I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an alpha-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu (I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu (I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600nm emission. It was further found that the exogenous reductant tris (2-carboxyethyl) phosphine (TCEP) can competitively inhibit the binding of Cu (I) to the low affinity site of the peptide, but does not interact with Cu (I) clusters. |
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