The Role of the Internal Disulfide Bond in the Conformational Dynamics of Neuroglobin

Abstract

Neuroglobin (Ngb) is a member of the globin family expressed mainly in brain tissue of mammals and other vertebrates that plays a role in the neuronal response to hypoxia and ischemia. Human Ngb has two cysteine residues (Cys46 and Cys55) within the CD loop and the D helix of the protein that were shown to form an internal disulfide bond, however these two residues are replaced by Gly in rat Ngb and the internal disulphide bond is missing. Therefore, to investigate the impact of the internal disulfide bond on the dynamics and energetics in Ngb we used photoacoustic calorimetry and transient absorption spectroscopy and determined the time-resolved volume and enthalphy changes associated with CO rebinding to human Ngb, rat Ngb and a rat Ngb mutant with and engineered internal disulfide bond (Cys46rNgb). The relaxation of the protein structure associated with the ligand photo-release is fast ( < 50 ns) and involves a decrease in the volume of the protein matrix. The enthalpy change associated to CO photo-dissociation for all samples studied was 19 kcal mol−1, whereas the reaction volume changes for human Ngb and Cys46rNgb were roughly two times higher than for rat Ngb. The reaction volume changes obtained for human and Cys46rNgb were 13.4±0.9 mL mol−1 and 10.3±0.6 mL mol−1, respectively, and 4.6±0.3 mL mol−1 for rat Ngb. These results indicate that the presence of the engineered internal disulphide bond in Cys46rNgb leads to a structural volume change that is similar to that found for human Ngb indicating that the internal disulphide bond control, to some extend, conformational dynamics associated with the ligand binding to deoxy Ngb.