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

色蛋白脱辅基蛋白识别和释放发色团的机制分析

• 批准号: 14380284
• 负责人: TANAKA Toshiyuki
• 金额: $ 9.6万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380284/
• 关键词: chromoprotein; antibiotics; molecular recognition; site-directed mutagenesis; NMR; calorimetry

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.

C-1027是最有效的抗肿瘤抗生素色蛋白之一，并且是具有DNA切割能力的烯二炔发色团和载体脱辅基蛋白的1：1复合物。本课题的目的是研究脱辅基蛋白对生色团的识别和释放机制，我们制备了脱辅基蛋白<13><15>C-1027及其与芳香化生色团的复合物，并测定了C-1027脱辅基蛋白的三维溶液结构。基于这些高分辨率的结构，我们能够阐明生色团和脱辅基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，我们进行了结合实验，通过量热法使用芳香化的生色团和一系列的突变脱辅基蛋白，其中的酸性，碱性或亲水性氨基酸残基被替换为不同类型的氨基酸。结合实验表明，Asp 101羧基与发色团18-N^+H_3之间的盐桥作用是最重要的静电相互作用，而His 104中邻近的咪唑环对这种相互作用有调节作用。我们还分析了骨架动力学的脱辅基蛋白在自由和结合状态，并能够指定的氨基酸残基，发挥重要作用，在释放的发色团。最后，我们成功地创造了突变蛋白质，它可以结合两个溴化乙锭分子，而天然的脱辅基蛋白质永远不会结合。