Structural and Functional Characterization of a Permuted Hemoglobin

The tetrameric composition of human hemoglobin complicates protein engineering efforts that are required to improve its potential as an oxygen-carrying therapeutic. In our research to design a single-chain version of the hemoglobin molecule (scHb), we have co-expressed a circularly permuted human β-globin (cp-β) with human α-globin. At micromolar concentrations, the purified recombinant globins appear to associate to form an α-cpβ heterodimer in solution rather than the expected α2-cpβ2 heterotetramer. Compared to recombinant human hemoglobin, the α-cpβ heterodimer exhibits a stronger ligand binding affinity. Knowledge of the intermolecular interactions favoring formation of the α-cpβ heterodimer will be instrumental in understanding the global structural consequences of the cpβ mutation, and in directing future protein engineering efforts to optimize the function of permuted hemoglobins. X-ray diffraction of α-cpβ crystals has been employed to determine the molecular structure of this protein complex at near-atomic resolution (2.7 Å). Examination of the structure shows that, in the crystal, the subunits associate to form a heterotetramer similar to that of wild-type hemoglobin in the high affinity R-state . The structure also confirms the incorporation of the β-globin His146 (wild-type numbering) into the cpβ linker, which removes an important ionic interaction that stabilizes the low oxygen affinity (T state) conformation. Structural information obtained as a result of this work will guide future protein engineering efforts to enhance cooperativity of oxygen binding and the T-state stability of future hemoglobin constructs