Studies on the structure and biochemical characteristics of the N-terminal and Globin domains of human Androglobin

Androglobin (Adgb) is a multidomain, circularly permuted protein discovered in 2012. To date, there have been little significant progress in its experimental characterisation due to the lack of expression of stable forms of the protein and its domains. Recently, an examination of the protein sequence alignments have enabled the successful expression of the globin domain (Adgb-GD) of androglobin. This thesis examined methods to optimize protein expression for producing a functional holo Adgb-GD protein suitable for biochemical analysis and characterisation. Biochemical characterisation revealed that Adgb is expressed predominantly as a hexacoordinate globin, both in the ferrous and ferric oxidation states, exhibiting optical characteristics similar to those of neuroglobin (Ngb) and cytoglobin (Cygb). However, the ferric state of Adgb exhibits pentacoordinate-like features, resembling the behaviour observed in Cygb, suggesting a potentially more complex coordination dynamics that is later explored in this study. The role of the disulfide bond in Adgb-GD ligand binding was also investigated. The findings demonstrate that the disulfide bond not only contributes to the stabilisation of the heme pocket by forming an ‘artificial’ CD loop typically absent due to the circular permutation of the Adgb globin domain but also plays a critical role in modulating ligand-binding behaviour, including potential interactions with other residues involved in protein-ligand interactions. Structural insights obtained from SEC-SAXS and SEC-UHPLC experiments, along with studies on the kinetics of ligand-binding, indicate that Adgb-GD exists as a heterogeneous population with multiple conformers. Moreover, Adgb-GD exhibited robust in vitro nitrite reductase activity, suggesting a potential redox regulation physiological role for the protein in vivo. To our knowledge, this is the first study to experimentally investigate the N-terminal, calpain-like region of Adgb. The findings support previous studies that suggest this region possesses autoproteolytic activity. Additionally, a novel nucleophilic cysteine site is proposed at Cys206, based on its high sequence conservation and the observation that mutation at this position enabled, for the first time, the successful expression of the full-length N-terminal region of the protein.