Kovalska Vladyslava, Vakarov Serhii, Losytskyy Mykhaylo, Kuperman Marina, Chornenka Nina, Toporivska Yuliya, Gumienna-Kontecka Elzbieta, Voloshin Yan, Varzatskii Oleg, Mokhir Andriy
Institute of Molecular Biology and Genetics, NASU 150 Zabolotnogo St. 03143 Kyiv Ukraine
Princeton Biomolecular Research Labs 26A Saperne Pole St. 01042 Kyiv Ukraine.
RSC Adv. 2019 Aug 5;9(42):24218-24230. doi: 10.1039/c9ra04102h. eCollection 2019 Aug 2.
Cage metal complexes iron(ii) clathrochelates, which are inherently CD silent, were discovered to demonstrate intensive output in induced circular dichroism (ICD) spectra upon their assembly to albumins. With the aim to design clathrochelates as protein-sensitive CD reporters, the approach for the functionalization of one chelate α-dioximate fragment of the clathrochelate framework with two non-equivalent substituents was developed, and constitutional isomers of clathrochelate with two non-equivalent carboxyphenylsulfide groups were synthesized. The interaction of designed iron(ii) clathrochelates and their symmetric homologues with globular proteins (serum albumins, lysozyme, β-lactoglobulin (BLG), trypsin, insulin) was studied by protein fluorescence quenching and CD techniques. A highly-intensive ICD output of the clathrochelates was observed upon their association with albumins and BLG. It was shown that in the presence of BLG, different clathrochelate isomers gave spectra of inverted signs, indicating the stabilization of opposite configurations ( or ) of the clathrochelate framework in the assembly with this protein. So, we suggest that the isomerism of the terminal carboxy group determined preferable configurations of the clathrochelate framework for the fixation in the protein binding site. MALDI TOF results show the formation of BLG-clathrochelate complex with ratio 1 : 1. Based on the docking simulations, the binding of the clathrochelate molecule (all isomers) to the main BLG binding site (calyx) in its open conformation is suggested. The above results point that the variation of the ribbed substituents at the clathrochelate framework is an effective tool to achieve the specificity of clathrochelate ICD reporting properties to the target protein.
笼形金属配合物铁（II）包结螯合物本身是圆二色性（CD）沉默的，然而人们发现，当它们与白蛋白组装时，在诱导圆二色性（ICD）光谱中会显示出强烈的信号输出。为了将包结螯合物设计成蛋白质敏感的CD报告分子，开发了一种用两个不等价取代基对包结螯合物骨架的一个螯合α - 二肟片段进行功能化的方法，并合成了带有两个不等价羧基苯硫醚基团的包结螯合物的构造异构体。通过蛋白质荧光猝灭和CD技术研究了设计的铁（II）包结螯合物及其对称同系物与球状蛋白质（血清白蛋白、溶菌酶、β - 乳球蛋白（BLG）、胰蛋白酶、胰岛素）的相互作用。当包结螯合物与白蛋白和BLG结合时，观察到了它们高度强烈的ICD信号输出。结果表明，在BLG存在的情况下，不同的包结螯合物异构体给出了相反符号的光谱，这表明在与该蛋白质组装时，包结螯合物骨架的相反构型（或）得到了稳定。因此，我们认为末端羧基的异构现象决定了包结螯合物骨架在蛋白质结合位点固定时的优选构型。基质辅助激光解吸电离飞行时间（MALDI TOF）结果表明形成了比例为1∶1的BLG - 包结螯合物复合物。基于对接模拟，推测包结螯合物分子（所有异构体）与处于开放构象的主要BLG结合位点（花萼）结合。上述结果表明，改变包结螯合物骨架上的肋状取代基是实现包结螯合物ICD报告特性对目标蛋白质特异性的有效手段。
