Abstract
Our study examines the functional and structural effects of amino acid substitution in the distal side of beta-chains of human Hb Duarte (alpha(2)beta(2)(62Ala-->Pro)). We have compared the functional properties of the purified Hb Duarte with those of HbA, and through proton NMR and molecular dynamics simulations we have investigated their tertiary and quaternary structures. The variant exhibits an increased oxygen affinity with a normal Hill coefficient and Bohr effect. The abnormal function of Hb Duarte is attributed to the presence of a proline residue at the beta62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (beta(62Ala-->Asp)), have been described as normal. Thereafter (1)H-NMR studies have shown that the beta62 Ala-->Pro substitution causes structural modifications of the tertiary structure of the beta globins, leaving the quaternary structure unaltered. These results have been confirmed by extensive all-atom molecular dynamics simulations. All these findings lead to the conclusion that the beta62 Ala-->Pro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the beta-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.