Bhagi-Damodaran A, Hosseinzadeh P, Mirts E, Reed J, Petrik I D, Lu Y
University of Illinois at Urbana-Champaign, Urbana, IL, United States.
University of Illinois at Urbana-Champaign, Urbana, IL, United States.
Methods Enzymol. 2016;580:501-37. doi: 10.1016/bs.mie.2016.05.050. Epub 2016 Jul 26.
Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.
异核金属酶催化自然界中一些最具根本意义且实用的反应。然而，这些酶中两个或更多金属离子紧密相邻的存在，使得它们比同核金属酶更难制备和研究。为应对这些挑战，异核金属中心已被设计到具有刚性支架的小而稳定的蛋白质中，以了解这些异核中心是如何构建的及其功能机制。本章通过给出在肌红蛋白和细胞色素c过氧化物酶中工程化的一些血红素 - 非血红素双金属中心的具体例子，描述了在蛋白质支架中设计异双核金属中心的方法。我们提供了关于如何选择蛋白质支架、通过计算在蛋白质支架中设计异双核金属中心、将金属离子掺入蛋白质以及从结构和功能上表征所得金属蛋白的分步程序。最后，我们讨论了如何通过合理调节其二级配位层、电子/质子转移速率和底物亲和力来进一步改进初始设计。
