An intramolecular disulfide bond designed in myoglobin fine-tunes both protein structure and peroxidase activity

Lei-Bin Wu; Hong Yuan; Hu Zhou; Shu-Qin Gao; Chang-Ming Nie; Xiangshi Tan; Ge-Bo Wen; Ying-Wu Lin

doi:10.1016/j.abb.2016.04.012

An intramolecular disulfide bond designed in myoglobin fine-tunes both protein structure and peroxidase activity

Arch Biochem Biophys. 2016 Jun 15:600:47-55. doi: 10.1016/j.abb.2016.04.012. Epub 2016 Apr 23.

Authors

Lei-Bin Wu¹, Hong Yuan², Hu Zhou³, Shu-Qin Gao⁴, Chang-Ming Nie¹, Xiangshi Tan², Ge-Bo Wen⁴, Ying-Wu Lin⁵

Affiliations

¹ School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
² Department of Chemistry, Shanghai Key Lab of Chemical Biology for Protein Research, Institute of Biomedical Science, Fudan University, Shanghai 200433, China.
³ Department of Analytical Chemistry, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
⁴ Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China.
⁵ School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China; Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China. Electronic address: [email protected].

PMID: 27117233
DOI: 10.1016/j.abb.2016.04.012

Abstract

Disulfide bond plays crucial roles in stabilization of protein structure and in fine-tuning protein functions. To explore an approach for rational heme protein design, we herein rationally introduced a pair of cysteines (F46C/M55C) into the scaffold of myoglobin (Mb), mimicking those in native neuroglobin. Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond, which was confirmed experimentally by ESI-MS analysis, DTNB reaction and CD spectrum. Moreover, it was shown that the spontaneously formed disulfide bond of Cys46-Cys55 fine-tunes not only the heme active site structure, but also the protein functions. The substitution of Phe46 with Ser46 in F46S Mb destabilizes the protein while facilitates H2O2 activation. Remarkably, the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and regulates the heme site to be more favorable for substrate binding, resulting in enhanced peroxidase activity. This study provides valuable information of structure-function relationship for heme proteins regulated by an intramolecular disulfide bond, and also suggests that construction of such a covalent bond is useful for design of functional heme proteins.

Keywords: Coordination state; Disulfide bond; Heme proteins; Peroxidase activity; Protein design.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Amino Acid Substitution
Binding Sites
Computer Simulation
Cysteine / chemistry
Disulfides / chemistry*
Models, Chemical
Models, Molecular
Molecular Sequence Data
Myoglobin / chemistry*
Myoglobin / ultrastructure*
Peroxidase / chemistry*
Peroxidase / ultrastructure*
Protein Binding
Protein Conformation
Protein Engineering / methods*
Structure-Activity Relationship

Substances

Disulfides
Myoglobin
Peroxidase
Cysteine