Strategy for Structural Characterization of Haemoglobin Variants

Baudin-Chich, V.; Rochette, Jacques; Wajcman, Henri

doi:10.1159/000205860

Cited by 16 publications

(8 citation statements)

References 4 publications

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“…Fluorescence studies suggest that tryptophan at the j?6 position is exposed on the surface of hemoglobin [16], and our results indicate that the presence of hydrophobic amino acid side chains on the surface may facilitate instability of hemoglobin. Understanding the structural basis of Hb S instability is important, since instability of oxy-Hb S is believed to be the primary cause of oxidant damage of the red cell membrane in sickle cell disease [29].…”

Section: Mechanical Stability Of Hemoglobins Mod$ed At the /I6 Positionmentioning

confidence: 60%

“…Previous studies show that deoxyhemoglobin solubility is lower with Ile and Leu at the 86 position than with Val, and that both hydrophobic substitutions accelerate polymerization [ 16,181. More recent studies show that tryptophan at the /?6 position does not promote tetramer polymerization, suggesting that there may be steric hindrance at the acceptor site of a lateral contact on polymers [ 171.…”

Section: Introductionmentioning

confidence: 93%

“…Recombinant DNA approaches promise to help de-47 fine the relationship between protein structure and function and to increase our knowledge of basic molecular mechanisms involved in polymerization and stability of hemoglobin [14-181. Previous studies show that deoxyhemoglobin solubility is lower with Ile and Leu at the 86 position than with Val, and that both hydrophobic substitutions accelerate polymerization [ 16,181. More recent studies show that tryptophan at the /?6 position does not promote tetramer polymerization, suggesting that there may be steric hindrance at the acceptor site of a lateral contact on polymers [ 171.…”

Section: Introductionmentioning

confidence: 93%

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Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

et al. 1993

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The relationship between different amino acids at the/l6 position of hemoglobin and tetramer stability was addressed by a site-directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu and Trp at the 86 position increased 2, 5, 13, 21 and 53 times, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the 86 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobic&y of/?6 Val. Deoxyhemoglobin solubility increased in the order of/36 Be, Leu, Val, Trp, Gln, Ala and Glu suggesting that hydrophobic interactions between 86 Val and the acceptor site of another hemoglobin molecule during deoxy-Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the 86 position. Furthermore, our results indicate that hydrophobic amino acids at the 86 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.

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Section: Mechanical Stability Of Hemoglobins Mod$ed At the /I6 Positionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 93%

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Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

et al. 1993

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“…The presence of the mutation was verified by DNA sequencing and protein analysis by reverse-phase high performance liquid chromatography (Baudin-Chich et al, 1987). Reassembled tetramers showed visible absorption spectra identical to those of native Hb A and Hb S in their deoxy and liganded forms.…”

Section: Resultsmentioning

confidence: 96%

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins

et al. 1992

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Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb S, 06 Glu + Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S 0 subunits in Escherichia coli provides the experimental tools to test these models. For H b S, the 06Val 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 hydrophobicity of the residue at the 06 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 06 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 023 Val -+ Ile mutation at the axial contact with either the 06 Glu -+ Val or 06 Glu + Ile substitution in the same 0 subunit. In comparison to native H b S the solubility of these deoxyHb double mutants is decreased two-and fourfold, respectively.

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“…The elution was obtained by developing a non-linear gradient of 5 -50% acetonitrile in 0.05% trifluoracetic acid in water with a flow rate of 1.5 ml/min. This system of solvent was found to be more suitable for the study of a-chain tryptic digest than the classical ammonium acetate system which, conversely, is more informative for the jl chains [9].…”

Section: Structural Studiesmentioning

confidence: 99%

Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [α88(F9)Ala→Ser]

Baklouti

Baudin-Chich

Kister

et al. 1988

European Journal of Biochemistry

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Increased homotropic allosteric effect, while maintaining normal heterotropic effects, was observed in hemoglobin Loire. The oxygen binding curves, at equilibrium, and the kinetic measurements demonstrated that the substitution of a88(F9) Ala for a Ser results in increased oxygen affinity and decreased ns0 value. The function of the residues involved in the Bohr effect or in the regulation by 2,3-bisphosphoglycerate is not altered. The effects of bezafibrate, which binds specifically to the a chains, was similar to that observed in Hb A. The functional properties of Hb Loire may be explained by a slight displacement of some key residues of the Cterminal region of the a chain destabilizing the T structure.

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Strategy for Structural Characterization of Haemoglobin Variants

Cited by 16 publications

References 4 publications

Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins

Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [α88(F9)Ala→Ser]

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