Robust affinity standards for Cu(I) biochemistry.
Robust affinity standards for Cu(I) biochemistry.
复制标题
DOI：
10.1021/ja408827d
复制
发表时间：
2013-12-11
影响因子：
15
通讯作者：
Fahrni CJ
中科院分区：
文献类型：
--
作者：
Bagchi P;Morgan MT;Bacsa J;Fahrni CJ
测量可靠的铜(I)蛋白结合亲和力需要具有相似结合强度的竞争参考配体；然而，关于这种参考配体的文献不仅稀少，而且经常相互冲突。为了解决这一不足，我们创造并表征了一系列水溶性的单价铜配体MCL-1、MCL-2和MCL-3，它们在水溶液中与铜(I)形成了明确的、空气稳定的无色络合物。从X-射线结构数据、电化学测量和广泛的平衡滴定网络得出结论，这三个配体都形成了化学计量比为1：1的离散的铜(I)络合物，能够缓冲10−10到10−17 M之间的铜(I)浓度。由于大多数铜(I)蛋白的亲和力是通过与二磺酸或2，2‘-二氨基丁酸的竞争实验获得的，我们进一步校准了它们与MCL系列的铜(I)稳定常数。为了证明这些试剂的应用，我们测定了CusF(logK=14.3±0.1)与铜(I)的结合亲和力，CusF是一种周质金属蛋白，用于解毒大肠杆菌中升高的铜水平。总之，这套相互关联的亲和力标准建立了可靠的基础，将有助于精确测定蛋白质和小分子配体的铜(I)结合亲和力。
The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. Concluding from X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations, all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10−10 and 10−17 M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate (BCS) or 2,2′-bicinchoninic acid (BCA), we further calibrated their Cu(I) stability constants against the MCL-series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (logK = 14.3±0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in E. coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small molecule ligands.
登录
查看更多内容
影响因子：
14.8
作者：
Fu, Yue;Tsui, Ho-Ching Tiffany;Bruce, Kevin E.;Sham, Lok-To;Higgins, Khadine A.;Lisher, John P.;Kazmierczak, Krystyna M.;Maroney, Michael J.;Dann, Charles E., III;Winkler, Malcolm E.;Giedroc, David P.
通讯作者：
Giedroc, David P.
影响因子：
2.9
作者：
Hansen, Lee D.;Fellingham, Gilbert W.;Russell, Donald J.
通讯作者：
Russell, Donald J.
影响因子：
15
作者：
Badarau, Adriana;Dennison, Christopher
通讯作者：
Dennison, Christopher
影响因子：
3.2
作者：
Franke, S;Grass, G;Nies, DH
通讯作者：
Nies, DH
影响因子：
15
作者：
DESPER, JM;GELLMAN, SH;COOPER, SR
通讯作者：
COOPER, SR