Analysis of the mechanisms for the recognition and the release of chromophores by chromoprotein apoproteins

C-1027是最有效的抗肿瘤抗生素色素蛋白之一，是具有dna切割能力的烯二炔发色团和载脂蛋白载体的1:1复合物。本项目旨在了解载脂蛋白识别和释放发色团的机制。我们制备了^<13>C/^<15> n标记的载脂蛋白，并测定了C-1027载脂蛋白及其与芳构化发色团配合物的三维溶液结构。基于这些高分辨率结构，我们能够阐明发色团和载脂蛋白之间可能的结合相互作用。为了证实可能的静电相互作用，我们使用芳香化的发色团和一系列突变载子蛋白进行了结合实验，其中酸性、碱性或亲水性氨基酸残基被不同类型的氨基酸取代。结合实验表明，最重要的静电相互作用是Asp101羧酸盐与发色团的18-N^+H_3之间的盐桥作用，而这种相互作用是由His104附近的咪唑环调制的。我们还分析了载脂蛋白在游离和结合状态下的骨架动力学，并能够指定在释放发色团中起重要作用的氨基酸残基。最后，我们成功地创造了能够结合两个溴化乙啶分子的突变蛋白，这是天然载脂蛋白无法结合的。

C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The purpose of this project is to understand the mechanisms for the recognition and the release of chromophore by the apoprotein.We prepared the ^<13>C/^<15>N-labelled apoprotein and determined the three-dimensional solution structure of C-1027 apoprotein and its complex with the aromatized chromophore. Based on these high-resolution structures, we were able to elucidate the possible binding interactions between the chromophore and the apoprotein. To confirm the possible electrostatic interactions, we performed the binding experiments by calorimetry using the aromatized chromophore and a series of mutant apoproteins, of which an acidic, basic, or hydrophilic amino acid residue is replaced with the different type of amino acids. From the binding experiments, we concluded that the most important electrostatic interaction is the salt bridge between the Asp101 carboxylate and the 18-N^+H_3 of the chromophore and that the interaction is modulated by the nearby imidazole ring of His104. We also analyzed the backbone dynamics of the apoprotein in both free and bound states and were able to specify the amino acid residues that play an important role in releasing the chromophore. Finally, we succeeded in creating the mutant proteins that could bind two molecules of ethidium bromide, which the natural apoprotein never binds.