Functional studies and polymerization of recombinant hemoglobin Glu-alpha2beta26(A3) --> Val/Glu-7(A4) --> Ala.
In hemoglobin (Hb) S the hydrophobic mutated residue Val-beta6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-beta85(F1) and Leu-beta88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The beta6(A3) residue is followed by two charged residues Glu-beta7(A4) and Lys-beta8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the beta7(A4) residue next to the donor site Val-beta6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb betaE6V/E7A). The single mutant Glu-beta7 --> Ala-(rHb betaE7A) 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 microM in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb betaE6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant betaE7A does not polymerize in the same experimental conditions. The Glu-beta7(A4) --> 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-beta7(A4) and Lys-beta132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.
Lesecq
S
S
Institut National de la Santé et de la Recherche Médicale, Unité 299, Hôpital de Bicêtre, 94275 Le Kremlin-Bicêtre, France.
Baudin
V
V
Kister
J
J
Marden
M C
MC
Poyart
C
C
Pagnier
J
J
D000409:Alanine; D003196:Computer Graphics; D005914:Globins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D006441:Hemoglobin A / Q000737:chemistry / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010108:Oxyhemoglobins / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D014633:Valine
D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D018698:Glutamic Acid; D005914:Globins; D006441:Hemoglobin A; D014633:Valine; D000409:Alanine