Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin
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- 作者：Xiao-Gang Shu ; Dr. Ji-Hu Su ; Dr. Ke-Jie Du ; Yong You ; Shu-Qin Gao ; Prof. Dr. Ge-Bo Wen ; Prof. Dr. Xiangshi Tan and Prof. Dr. Ying-Wu Lin
- 刊名：ChemistryOpen
- 出版年：2016
- 出版时间：June 2016
- 年：2016
- 卷：5
- 期：3
- 页码：192-196
- 全文大小：1335K
- ISSN：2191-1363
文摘
Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper-binding site of [3-His] or [2-His-1-Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non-heme proteins.