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 (KAH), G80A/H81A (KAH), and G80A/H81A/H82A (KA). 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 KAH, KAH, and KA variants, respectively, with the KAH Mb dimer being stabilized by a hydrogen bond network at the hinge region. When expressed and purified from , the dimer ratio increased in the order WT (1 ± 1%) < KAH (16 ± 3%) < KAH (35 ± 1%) < KA (82 ± 5%). A similar order was observed for the dimer ratio obtained upon reconstitution from apo Mb. The apo KA 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 KA 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.