结合金属卟啉辅助因子的四螺旋束蛋白的重新设计。

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-14 DOI:10.1016/bs.mie.2025.06.038
Karen R Coronado, Yixuan Zhu, Samuel I Mann
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引用次数: 0

摘要

自然界中血红素蛋白的多功能性源于其蛋白质支架的复杂控制。从头开始的蛋白质设计提供了一种强大的手段来解剖和重建这些结构功能关系,从而能够构建具有定制功能的新型金属蛋白。在这里,我们描述了MPP1的计算设计和表征,MPP1是一种四螺旋束蛋白,旨在结合一种非生物mn -二苯基卟啉(MnDPP)辅因子。利用参数化线圈骨架、Rosetta中灵活的骨架序列设计和结构引导的环路构建,MPP1被设计成具有轴向配体和第二壳相互作用精确定位的辅助因子,以及有目的的氧化剂和底物可达性。所得蛋白是第一个晶体学表征从头设计卟啉结合蛋白。MPP1表现出稳定高价Mn(V)-氧物质和介导硫醚氧化的能力。本章详细介绍了设计和评估能够以原子水平精度结合卟啉和卟啉样辅因子的四螺旋束蛋白所需的计算策略、辅因子结合和溶液表征。关键词:从头设计,蛋白质设计,生物无机化学,金属卟啉,血红素蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
De novo design of four-helix bundle proteins to bind metalloporphyrin cofactors.

The versatility of heme proteins in nature stems from the intricate control exerted by their protein scaffolds. De novo protein design offers a powerful means to dissect and recreate these structure-function relationships, enabling construction of novel metalloproteins with tailored functionalities. Here, we describe the computational design and characterization MPP1, a four-helix bundle protein designed to bind an abiological Mn-diphenylporphyrin (MnDPP) cofactor. Using parameterized coiled-coil backbones, flexible backbone sequence design in Rosetta, and structure-guided loop building, MPP1 was designed to accommodate the cofactor with precise positioning of axial ligands and second-shell interactions, as well as purposeful accessibility for oxidants and substrates. The resulting protein was the first crystallographically characterized de novo designed porphyrin-binding protein. MPP1 demonstrated the ability to stabilize a high-valent Mn(V)-oxo species and mediate thioether oxidation. This chapter details the computational strategies, cofactor incorporation, and solution characterization necessary to design and evaluate four-helix bundle proteins capable of binding porphyrin and porphyrin-like cofactors with atomic-level precision. Keywords: de novo design, protein design, bioinorganic chemistry, metalloporphyrins, heme proteins.

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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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