Abstract
Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb S, Ξ²6 Glu β Val) have been obtained from Xβray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Siteβdirected mutagenesis with expression of the Hb S Ξ² subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the Ξ²6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophoβbicity of the residue at the Ξ²6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the Ξ²36 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the Ξ²23 Val β Ile mutation at the axial contact with either the Ξ²6 Glu β Val or Ξ²6 Glu β Ile substitution in the same Ξ² subunit. In comparison to native Hb S the solubility of these deoxyHb double mutants is decreased twoβ and fourfold, respectively.