化学合成法构建基于蛋白质Nitrophorin-2的人工金属酶

人工金属酶是一类杂合催化剂，它结合了酶催化及金属催化的优点，已被用于多类重要非天然反应的催化，在发展绿色、可持续催化合成方法研究中受到广泛关注。然而，以共价偶联修饰为代表的人工金属酶传统构建方式多存在修饰位点受限、选择性欠佳等弊端；基于基因密码子扩展技术的人工金属酶可一定程度地解决上述问题，但相关技术门槛仍较高。结合申请人在金属蛋白质及其化学合成技术的研究积累，本项目拟探索化学合成法高效构建人工金属酶。以课题组熟悉的Nitrophorin为模板蛋白骨架，通过化学合成法选择性地引入多种含氮金属螯合配体，利用EPR等波谱学手段研究其与Cu(II)离子的结合，并将其用于非对称Diels-Alder反应的催化研究。利用分子对接、谱学表征及晶体结构解析等对其机理进行深入探讨，以对人工酶分子的再设计提供指导。本项目的顺利实施有助于进一步发展修饰蛋白质的制备技术，提供一种人工金属酶构建的新型通用方法。

Artificial metalloenzyme (ArMs) is a type of hybrid catalyst, combing the advantages of both enzymatic catalysis and organometallic catalysis, which has been applied for the transformation of many non-natural reactions. As such, ArMs have been at the forefront of researches developing more greener and sustainable catalytic reactions. The traditional means for the construction of ArMs, such as covalent conjugation, however, suffer from drawbacks like limited available modification site and low selectivity; ArMs based on the recently developed genetic code expansion technology solved the issue to a certain extent, but its technical requirements are still too high. Based on out previous research on metalloprotein and its chemical synthesis, we aim to develop an effective method constructing ArMs through chemical protein synthesis. Using nitrophorin that has been intensively investigated in our group as a model protein scaffold, we plan to insert the non-natural metal ligand sidechains via chemical synthesis. We will probe the metal (CuII) coordination process via EPR and related spectroscopic methods, more importantly, investigate the catalytic performance of the synthetic protein in catalyzing the non-symmetric Diels-Alder reaction. Through molecular docking, spectroscopy and X-ray crystallography, we hope to understand the catalytic mechanism, which may set the stage for further design of advanced ArMs. The realization of this proposal will further advance our techniques for protein modification and provide a conceptually novel and general method for the efficient construction of ArMs.