Department of Chemical Sciences, University of Naples Federico II, Via Cintia, 80126, Naples, Italy.
Department of Biotechnology and Biosciences, University of Milan-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
Angew Chem Int Ed Engl. 2023 Jan 2;62(1):e202211552. doi: 10.1002/anie.202211552. Epub 2022 Nov 29.
De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.
从头设计金属蛋白是一种将蛋白质支架塑造成特定功能的卓越方法。在这里，我们报告了 Due Rame 1（DR1）的设计和表征，这是一种从头设计的蛋白，其中包含一个二铜位点，模拟了第 3 型（T3）含铜多酚氧化酶（PPO）。为了实现这一目标，我们分层设计了第一个和第二个双金属配位球，将二铜位点工程化为一个简单的四螺旋束支架。光谱、热力学和功能表征表明，DR1再现了 T3 铜位点，支持不同的铜氧化还原态，并能在 O 依赖性的儿茶酚氧化为邻醌中发挥作用。底物进入位点残基的精心设计赋予了 DR1 对底物的识别能力，这一点通过哈米特分析和对取代儿茶酚的计算研究得到了揭示。这项研究代表了在设计的四螺旋束蛋白中构建功能性 T3 铜位点的一个重要范例。
