Cu(I) binding properties of a designed metalloprotein.
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.
Xie
Fei
F
Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Sutherland
Duncan E K
DE
Stillman
Martin J
MJ
Ogawa
Michael Y
MY
D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D003300:Copper; D007202:Indicators and Reagents; D008667:Metalloproteins; D010455:Peptides; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine