title: Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.
abstract: 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.
authors.affiliation: School of Chemistry and Chemical Engineering University of South China Hengyang 421001 P. R. China.
authors.forename: Xiao-Gang
authors.initials: XG
authors.lastname: Shu
authors.affiliation: Department of Modern Physics University of Science and Technology of China Hefei 230026 P. R. China.
authors.forename: Ji-Hu
authors.initials: JH
authors.lastname: Su
authors.affiliation: School of Chemistry and Chemical Engineering University of South China Hengyang 421001 P. R. China.
authors.forename: Ke-Jie
authors.initials: KJ
authors.lastname: Du
authors.affiliation: Laboratory of Protein Structure and Function University of South China Hengyang 421001 P. R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: You
authors.affiliation: Laboratory of Protein Structure and Function University of South China Hengyang 421001 P. R. China.
authors.forename: Shu-Qin
authors.initials: SQ
authors.lastname: Gao
authors.affiliation: Laboratory of Protein Structure and Function University of South China Hengyang 421001 P. R. China.
authors.forename: Ge-Bo
authors.initials: GB
authors.lastname: Wen
authors.affiliation: Department of Chemistry Shanghai Key Lab of Chemical Biology for Protein Research & Institute of Biomedical Science Fudan University Shanghai 200433 P. R. China.
authors.forename: Xiangshi
authors.initials: X
authors.lastname: Tan
authors.affiliation: School of Chemistry and Chemical Engineering University of South China Hengyang 421001 P. R. China; Laboratory of Protein Structure and Function University of South China Hengyang 421001 P. R. China.
authors.forename: Ying-Wu
authors.initials: YW
authors.lastname: Lin
keywords: electron paramagnetic resonance (EPR); heme proteins; metal-binding site; nitrite reductase; protein design