Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins.
Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins.
复制标题
用于设计蛋白质的人工金属依赖性调节位点的构象研究。
DOI：
10.1002/zaac.201300131
复制
发表时间：
2013
影响因子：
1.4
通讯作者：
Oheix E
中科院分区：
文献类型：
--
作者：
Oheix E
本报告描述了谷胱甘肽的二聚化，以及其他含半胱氨酸的肽或蛋白质片段，与 5, 5'-二取代-2, 2'-联吡啶或 6, 6''-二取代-2, 2':6',2''-三联吡啶单元的二聚化。所得的 bipy-GS2 和 terpy-GS2 在水溶液中作为潜在的金属离子依赖性开关进行了研究，并发现在生理 pH 下主要采用反式构象。在此 pH 值下金属与 CuII 和 ZnII 的络合已通过 UV/Vis、CD、NMR 和离子淌度质谱法进行了研究。 ZnII 滴定与 pH 7.4 下 1:1 ZnII:terpy-GS2 复合物的形成一致，但 bipy-GS2 显示可形成 1:1 和 1:2 复合物，在稀微摩尔条件下前者占主导地位。所得 1:1 复合物的形成常数确定为 logKM6.86 (bipy-GS2) 和 6.22 (terpy-GS2)，这与与受约束的三联吡啶相比，不受约束的联吡啶具有更高的亲和力一致。 CuII 配位涉及 1:1 配合物的初始形成，然后是微摩尔浓度的 1.5Cu:1bipy-GS2 和 2Cu:1terpy-GS2 配合物。形成 1:1 复合物的结合常数（logKM12.5 (bipy-GS2)；8.04 和 7.14 (terpy-GS2)）表明对 CuII 的亲和力高于 ZnII。最后，离子淌度质谱研究检测到质子化形式的游离配体，并且与气相中两种具有不同构象的不同铜加合物的形成一致。我们说明联吡啶和三联吡啶二聚单元可以像响应铜/锌络合的构象开关一样表现，并提出未来这些可以用于具有更大肽或蛋白质片段的合成生物学，以控制大规模折叠和相关生物功能。
This report describes the dimerisation of glutathione, and by extension, other cysteine‐containing peptides or protein fragments, with a 5, 5'‐disubstituted‐2, 2'‐bipyridine or 6, 6''‐disubstituted‐2, 2':6',2''‐terpyridine unit. The resultingbipy‐GS2andterpy‐GS2were investigated as potential metal ion dependent switches in aqueous solution, and were found to predominantly adopt thetransoïdconformation at physiological pH. Metal complexation with CuIIand ZnIIat this pH has been studied by UV/Vis, CD, NMR and ion‐mobility mass spectrometry. ZnIItitrations are consistent with the formation of a 1:1 ZnII:terpy‐GS2complex at pH 7.4, butbipy‐GS2was shown to form both 1:1 and 1:2 complexes with the former being predominant under dilute micromolar conditions. Formation constants for the resulting 1:1 complexes were determined to be logKM6.86 (bipy‐GS2) and 6.22 (terpy‐GS2), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. CuIIcoordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy‐GS2and 2Cu:1terpy‐GS2complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (logKM12.5 (bipy‐GS2); 8.04 and 7.14 (terpy‐GS2)) indicate a higher affinity for CuIIthan ZnII. Finally, ion‐mobility MS studies detected the free ligands in their protonated form, and were consistent with the formation of two different Cu adducts with different conformations in the gas‐phase. We illustrate that the bipyridine and terpyridine dimerisation units can behave like conformational switches in response to Cu/Zn complexation, and propose that in future these can be employed in synthetic biology with larger peptide or protein fragments, to control large scale folding and related biological function.