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Abstract
Hb S variants containing Lys-beta132 --> Ala or Asn substitutions were engineered to evaluate the consequences of the A helix destabilization in the polymerization process. Previous studies suggested that the loss of the Glu-beta7-Lys-beta132 salt bridge in the recombinant Hb betaE6V/E7A could be responsible for the destabilization of the A helix. The recombinant Hb (rHb) S/beta132 variants polymerized with an increased delay time as well as decreased maximum absorbance and Hb solubility values similar to that of Hb S. These data indicate that the strength of the donor-acceptor site interaction may be reduced due to an altered conformation of the A helix. The question arises whether this alteration leads to a true inhibition of the polymerization process or to qualitatively different polymers. The oxygen affinity of the beta132 mutated rHbs was similar to that of Hb A and S, whereas the cooperativity and effects of organic phosphates were reduced. This could be attributed to modifications in the central cavity due to loss of the positively charged lysine. Since Lys-beta132 is involved in the stabilization of the alpha1-beta1 interface, the loss of the beta132(H10)-beta128(H6) salt bridge may be responsible for the marked thermal instability of the beta132 mutated rHbs.
Highlights
The capacity of some Hb variants to facilitate or impair the polymerization process of Hb S is well documented [4]
We have previously demonstrated that the association of the E7A and E6V mutations on the same  chain leads to a decreased polymer formation; the Glu-7(A4) for Ala substitution in human Hb resulted in heat instability and in an increased oxygen affinity of the recombinant Hb (rHb) E7A and E6V/E7A [11]
In human Hbs A and S, Glu-7(A4) forms an intrachain salt bridge with Lys-132(H10) in both R- and T-state structures [24]. The loss of this salt bridge may modify the conformation of the A helix, which could account for the alteration of the polymerization process
Summary
The capacity of some Hb variants to facilitate or impair the polymerization process of Hb S is well documented [4]. Studies of recombinant Hbs (rHbs) have shown that in Hb S Antilles the polymer fibers were stabilized at the axial contact by the replacement of Val with the more hydrophobic residue Ile [10]. We have previously reported studies of the function and polymerization of another rHb, E6V/E7A [11] In this rHb, the association of Glu-6(A3) 3 Val and Glu-7(A4) 3 Ala mutations on the same  chain (rHb E6V/E7A) results in an apparent decrease of the polymer formation. Modification of the second partner in the salt bridge (Lys-132(H10)) may result in its rupture This residue participates in several contacts at the ␣1-1 interface [12] and might be involved in the stability of the A helix. We have engineered the single mutant Hbs Lys-␣22132(H10) 3 Ala and Lys-␣22132(H10) 3 Asn as controls (rHbs K132A and K132N, respectively)
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