Fig. 3.
Amino acid replacements at β13 and β83 produce second-order perturbations of tertiary structure that affect the allosteric regulation of O2-binding by IHP. (A) Homology-based structural model of hummingbird Hb showing the locations of amino acid replacements at sites 13 and 83 in the β1 subunit (shown in cyan). β-Chain residues 18–22, 42–45, and 81–84 (including the variable site 83, the penultimate C-terminal residue in the EF interhelical loop) alternate between helical and nonhelical secondary structure in the allosteric transition between the oxy (B) and deoxy (C) states. (D) Structural model of hummingbird Hb (with the β1 subunit removed) showing alternative IHP-binding sites in the central cavity. Polarity-changing amino acid replacements at β13 and β83 produce nonadditive changes in the favorability of alternative conformation states for polyphosphate-binding: IHP preferentially binds at “site 1” in the β13Gly-β83Gly and β13Ser-β83Ser mutants (representing wild-type β-globin genotypes for A. melanogenys and O. estella, respectively) and at “site 2” in the β13Ser-β83Gly and β13Gly-β83Ser mutants.