Apoprotein Intermolecular Interactions and Heme Insertion for 3D Domain Swapping in Myoglobin

Many proteins, including heme proteins undergo three-dimensional domain swapping (3D-DS). The loop between E and F helices is converted to a helical structure in the myoglobin (Mb) 3D-DS dimer. However, the relationship between 3D-DS and heme insertion in Mb remains unclear. Here, we systematically investigated the 3D-DS propensity of wild-type (WT) Mb and its variant in which one to three Ala residues were introduced into the hinge region: G80A (K₃AH₂), G80A/H81A (K₃A₂H), and G80A/H81A/H82A (K₃A₃). After heating the Mb monomer at 70 °C for 30 min, no dimers were detected for WT Mb, whereas dimers were formed by 55 ± 1%, 92 ± 2%, and 84 ± 2% of the protein molecules for the K₃AH₂, K₃A₂H, and K₃A₃ variants, respectively, with the K₃A₂H Mb dimer being stabilized by a hydrogen bond network at the hinge region. When expressed and purified from <i>Escherichia coli</i>, the dimer ratio increased in the order WT (1 ± 1%) < K₃AH₂ (16 ± 3%) < K₃A₂H (35 ± 1%) < K₃A₃ (82 ± 5%). A similar order was observed for the dimer ratio obtained upon reconstitution from apo Mb. The apo K₃A₃ Mb dimer exhibited higher helical propensities than its monomer and apo forms of the other variants. Molecular dynamics studies supported the hypothesis that the stabilization of the α-helices at the hinge region enhances dimer formation in K₃A₃ Mb compared to WT Mb and other variants. These results indicate that the formation of Mb 3D-DS dimers in vivo depends on the apo monomer-dimer equilibrium before heme insertion, showing that 3D-DS is significantly influenced by protein-folding conditions.