Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
J Inorg Biochem. 2010 Mar;104(3):261-7. doi: 10.1016/j.jinorgbio.2009.12.005. 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.
报告了设计的肽 C16C19-GGY 与 Cu(I) 的结合特性。该肽旨在形成α-螺旋卷曲螺旋，但经过修饰以在其疏水面上包含 Cys-X-X-Cys 金属结合基序。吸收、发射、电喷雾电离质谱 (ESI-MS) 和圆二色性 (CD) 实验表明，当 Cu(I) 最初添加到单体肽的溶液中时，形成了 1:1 Cu-肽复合物。这与我们之前的研究一致，其中显示发荧光的 1:1 配合物作为包含四核铜簇的四聚体肽存在 Kharenko 等人。(2005) [11]。ESI-MS 现在证实了四核铜中心的存在，与 UV 数据一起表明该簇以协同方式形成。然而，光谱滴定表明，继续添加 Cu(I) 会导致金属肽中第二个低亲和力金属结合位点的占据。这种占据不会显著影响金属肽的构象，但会导致 600nm 发射的猝灭。进一步发现，外源还原剂三(2-羧乙基)膦 (TCEP) 可以竞争性抑制 Cu(I)与肽的低亲和力位点的结合，但不与 Cu(I)簇相互作用。
