Functional Studies and Polymerization of Recombinant Hemoglobin Glu-α2β26(A3) → Val/Glu-7(A4) → Ala

Abstract

In hemoglobin (Hb) S the hydrophobic mutated residue Val-␤6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-␤85(F1) and Leu-␤88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The ␤6(A3) residue is followed by two charged residues Glu-␤7(A4) and Lys-␤8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the ␤7(A4) residue next to the donor site Val-␤6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb ␤E6V/E7A). The single mutant Glu-␤7 3 Ala-(rHb ␤E7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 M in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb ␤E6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant ␤E7A does not polymerize in the same experimental conditions. The Glu-␤7(A4) 3 Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-␤7(A4) and Lys-␤132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.

The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal

“…EBBP has glutamic acid residues within close proximity to both the N and C termini, so it is conceivable that some proteolytic processing might alter its net charge and cause a cathodic shift. However, anomalous behavior during isoelectric focusing of proteins has also been reported in the case of hemoglobin E7A and E6V/ E7A mutants (48). This kind of anomaly may also be due to the local effects of surrounding amino acids altering the theoretical pI or changes in protein-protein noncovalent interactions (49).…”

Influence of the A Helix Structure on the Polymerization of Hemoglobin S

“…Studies of recombinant Hbs (rHbs) 1 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.…”

“…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. We therefore postulated that this decrease could be due to an instability of the A helix because of the loss of a salt bridge between the A and H helices, namely between the Glu-␤7(A4) and Lys-␤132(H10) residues (11). Modification of the second partner in the salt bridge (Lys-␤132(H10)) may also result in its rupture.…”

β7E–β132K salt bridge and sickle haemoglobin stability and conformation

“…Recombinant haemoglobins (rHb) with substitutions that disrupt the β7 (A helix)–β132 (H helix) salt bridge have been investigated (Lesecq et al , 1996, 1997). β7 (A4) Glu forms an intrachain salt bridge with the β132 (H10) Lys in both the R‐ (liganded) and T‐ (deoxy) forms.…”

“…However, the cooperativity and effects of organic phosphates were reduced (Lesecq et al , 1996, 1997). Thus, the loss of the β7 (A4) Glu–β132 (H10) salt bridge might lead to an alteration in both the position and the mobility of the A‐helix that would change its polymerization and functional properties (Lesecq et al , 1996, 1997). The goal of the present study was to define the role of the β7–β132 salt bridge in Hb conformational integrity and stability.…”