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Structure-Function Relationship in a Variant Hemoglobin: A Combined Computational-Experimental Approach

Matteo Ceccarelli ^*, Paolo Ruggerone ^* , Roberto Anedda , Antonella Fais , Benedetta Era , Maria Carla Sollaino , Marcella Corda  and Mariano Casu 

^* CNR-INFM SLACS, Dipartimento di Fisica, Dipartimento di Scienze Chimiche, Dipartimento di Scienze Applicate ai Biosistemi Università di Cagliari, I-09042 Monserrato, Italy; and CNR-INFM CRS DEMOCRITOS, SISSA, I-34014 Trieste, Italy

Correspondence: Address reprint requests to Paolo Ruggerone, E-mail: paolo.ruggerone{at}dsf.unica.it.

Our study examines the functional and structural effects of amino acid substitution in the distal side of ß-chains of human Hb Duarte (). 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 ß62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (ß^62AlaAsp), have been described as normal. Thereafter ¹H-NMR studies have shown that the ß62 AlaPro substitution causes structural modifications of the tertiary structure of the ß 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 ß62 AlaPro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the ß-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.