Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala

Llano, José Javier Martı́n de; Manning, James M.

doi:10.1002/pro.5560030806

Cited by 38 publications

(16 citation statements)

References 33 publications

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“…Their results suggest that the relative order for polymerization of the b85 mutants of Hb S depends on amino acid hydrophobicity (Leu > Phe > Ala > Thr > Trpb85). Using a similar yeast-expression system, de Llano & Manning (1994) investigated the role of b88Leu in Hb S polymerization by constructing a mutant of Hb S, r Hb (b6Glu : Val, b88Leu : Ala). They found that the strength of the hydrophobic interaction between b6Val of one Hb S and b88Leu on an adjacent Hb S (i.e.…”

Section: Discussionmentioning

confidence: 99%

Roles of α114 and β87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy

Willis

Shen

et al. 1996

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Roles of α114 and β87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy

Willis

Shen

et al. 1996

Journal of Molecular Biology

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“…The three Hb S variants were subjected to DNA sequence analysis of the entire ␤-globin cDNA using site-specific primers and fluorescently tagged terminators in a cycle-sequencing reaction in which extension products were analyzed on an automated DNA sequencer (7,9). The mutated ␤-globin cDNA regions were then excised by XhoI digestion and subcloned back into the XhoI site of the expression vector pGS389 (4).…”

Section: Methodsmentioning

confidence: 99%

Role of Hydrophobic Amino Acids at β85 and β88 in Stabilizing F Helix Conformation of Hemoglobin S

Reddy

Surrey

et al. 1996

Journal of Biological Chemistry

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Three Hb S variants containing Glu substitutions at Phe-␤85 and/or Leu-␤88 were expressed in yeast in an effort to evaluate the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S. Helix stability of tetrameric Hb S ␤F85E,␤L88E was measured by CD and compared with those of Hb S ␤F85E, Hb S ␤L88E, Hb A, and Hb S. The CD spectra of these Hb S variants were similar to those of Hb S and Hb A at 10°C. However, changes in ellipticity at 222 nm for Hb S ␤F85E in the CO form at 60°C were about 15-fold greater than that of Hb S, while those for Hb S ␤L88E and Hb S ␤F85E,␤L88E were similar and about 30-fold greater than Hb S. Thermal stability measured by continuous scanning of spectral changes revealed the three Hb S variants were much more unstable than Hb S, and stability of Hb S ␤F85E,␤L88E was similar to that of Hb S ␤L88E rather than Hb S ␤F85E. These results suggest that Glu insertion at both ␤85 and ␤88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S ␤F85E,␤L88E is similar to Hb S ␤L88E rather than Hb S ␤F85E. Furthermore, these results suggest that both Phe-␤85 and Leu-␤88 are critical for F helix stabilization and that Glu insertion at ␤88 leads to more destabilization than insertion at ␤85.In addition to Val-␤6, the key to polymerization of deoxy-Hb S is formation of a Val-␤6 hydrophobic acceptor pocket containing the hydrophobic amino acids Phe-␤85 and Leu-␤88 in the F helices of the T structure or deoxy form of Hb S which interacts with Val-␤6 (1, 2). Therefore, characterization of the Val-␤6 acceptor site, which includes Phe-␤85 and Leu-␤88, is important to understand hydrophobic interactions of deoxy-Hb S polymerization.We previously engineered and expressed Hb S variants containing Glu at either ␤85 or ␤88, Hb S ␤F85E, and Hb S ␤L88E and characterized their polymerization properties in order to understand the role of hydrophobicity and stereospecificity of the acceptor pocket during deoxy-Hb S polymerization (3). The deoxy form of Hb S ␤L88E polymerized in vitro at a much higher hemoglobin concentration than deoxy-Hb S containing other amino acid substitutions at ␤85 and ␤88 such as Hb S ␤F85E and Hb S ␤L88F (3). Furthermore, kinetics of polymerization for Hb S ␤L88E were biphasic at lower hemoglobin concentrations. Deoxy-Hb S ␤F85E also polymerized like deoxy-Hb S ␤L88E; however, the concentration required for polymerization of Hb S ␤F85E was 3-fold less than that of Hb S ␤L88E.These differences in polymerization between having Glu at ␤85 versus ␤88 may be due to residue location in the acceptor pocket. X-ray analysis of crystallized deoxy-Hb S shows that ␤85 and ␤88 amino acid side chains line the bottom of the acceptor pocket (1, 2), with Phe-␤85 located internally at the floor of the pocket and Leu-␤88 located near the pocket entrance. This difference in location might be expected to lead to different results when making the same Glu change at these two sites. Polymerization properties of several other recombinan...

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“…Refinements in our knowledge of the mechanisms of polymerization of deoxyHb S and the effects of molecular and cellular alterations that might prove useful in treating sickle cell disease have become possible with the advent of techniques such as site-directed mutagenesis for preparing selected mutant Hbs (13,14), recombinant Hbs having both the Val-␤6 (sickle) substitution and another chosen substitution (double mutants) (15), as well as the development of varieties of sickling transgenic mice (16,17). In each case, it is important to assess accurately the polymerization and sickling tendency obtained with the new Hb or mixture, yet these Hbs are generally available in very limited amounts.…”

Section: Discussionmentioning

confidence: 99%

Polymer Structure and Solubility of Deoxyhemoglobin S in the Presence of High Concentrations of Volume-excluding 70-kDa Dextran

Bookchin

Balazs

Wang

et al. 1999

Journal of Biological Chemistry

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Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("C sat ") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the C sat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent C sat ("dextran-C sat ") was obtained together with acceptable sensitivity and proportionality with the standard C sat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70 -75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-tocrystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-C sat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.The solubility of deoxyHb 1 S polymers at equilibrium with the tetrameric "monomers" is a basic thermodynamic property of the polymerization process that underlies red cell sickling. Measurement of the polymer solubility, "C sat "(1) (also termed C s (2) or C sol (3)), of Hb S in conditions similar to those within the cells has served as a critical estimate of the relative polymerization tendency when comparing the native or modified Hb S, alone or in mixtures with non-S Hbs or inhibitors of polymerization. A concentrated solution of Hb S is fully deoxygenated to form a gel, and after high speed centrifugation to sediment the polymerized solid phase, the concentration of soluble Hb in the supernatant is the measured C sat .It is well documented that volume exclusion by macromolecules admixed with proteins results in their increased activity and corresponding interactions (4). Thus, when protein macromolecules capable of interacting, such as deoxyHb S, occupy a substantial fraction of the total volume of their medium, the addition of a second species of noninteracting macromolecules crowds the protein molecules closer together, increasing their interactions. More specifically, addition of macromolecules that do not copolymerize with deoxyHb S reduces its polymer solubility by this mechanism (5, 6). However, for the use of such observations in further investigations on the properties of deoxyHb S polymers, it is first necessary to characterize the morphology and properties of the polymer fibers generated in volume-excluded domains by comparison with those previously characterized for standard conditions.In the course of our ...

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Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala

Cited by 38 publications

References 33 publications

Roles of α114 and β87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy

Roles of α114 and β87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy

Role of Hydrophobic Amino Acids at β85 and β88 in Stabilizing F Helix Conformation of Hemoglobin S

Polymer Structure and Solubility of Deoxyhemoglobin S in the Presence of High Concentrations of Volume-excluding 70-kDa Dextran

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