Mutational analysis of phenylalanine β85 in the valine β6 acceptor pocket during hemoglobin S polymerization

Adachim, Kazuhiko; Reddy, Lattupally R.; Surrey, Saul; Reddy, Konda S.

doi:10.1002/pro.5560040703

Cited by 11 publications

(13 citation statements)

References 21 publications

(8 reference statements)

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“…Our results are consistent with the findings for the yeast-expressed r Hb (b6Glu : Val, b87Thr : Gln), which exhibits a prolonged delay time and inhibits polymerization (Adachi et al, 1994). Adachi et al (1995) have investigated the effect of the size of the amino acid residue located at b85 (i.e. in the b6Val acceptor pocket) by constructing four mutants of Hb S, with the replacement of b85Phe by Leu, Ala, Thr, or Trp.…”

Section: Discussionmentioning

confidence: 99%

“…Leu : Ala, at b88. Based on available data, Adachi et al (1995) have concluded that the stereospecificity of the b88 amino acid is more critical than that of b85 for inserting b6Val into the Hb S polymer. In addition, Manning and co-workers (Himanen et al, 1995) have investigated the participation and strength of interactions of b95Lys in Hb S polymerization by constructing a mutant of Hb S, i.e.…”

Section: Discussionmentioning

confidence: 99%

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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%

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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“…In addition, changes at ␤88 are also expected to increase oxygen affinity and decrease tetramer stability. In fact, our previous studies with the hydrophilic substitutions of Glu for both Phe-␤85 and Leu-␤88 showed increased oxygen affinity and decreased tetramer stability (3,6).…”

Section: Figmentioning

confidence: 94%

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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“…The polymerization-impairing or -enhancing propensity of mutant hemoglobins, in a binary mixture of mutant hemoglobins and HbS, has facilitated the mapping of several contact residues of the HbS polymer (10 -12). The list of contact sites has been expanded by subsequent studies involving chemical modifications of HbS (13,14) and site-directed mutagenesis (15)(16)(17)(18)(19). However, the identities of all the fiber contacts that are predicted by model studies have not yet been tested in solution experiments.…”

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confidence: 99%

A Role for the α113 (GH1) Amino Acid Residue in the Polymerization of Sickle Hemoglobin

Sivaram

Sudha

Roy

2001

Journal of Biological Chemistry

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A cluster of amino acid residues located in the AB-GH region of the ␣-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, ␣Leu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (␤Glu-63 Val, ␣Leu-1133 His) to ascertain the hitherto unknown role of the ␣113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C sat ) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the ␣113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the ␣113 mutation was coupled individually with two established fiber contact sites (␣16 and ␣23) located in the AB region of the ␣-chain: HbS(␣Lys-163 Gln, ␣Leu-1133 His), HbS(␣Glu-233 Gln, ␣Leu-1133 His). The single mutants at ␣16/␣23 sites were also engineered as controls. The C sat values of the HbS point mutants involving sites ␣16 or ␣23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C sat values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of ␣113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.Sickle cell anemia is a consequence of a point mutation (Glu-63 Val) at the sixth position in the ␤-chain of the hemoglobin molecule (1). The replacement of a charged residue with a hydrophobic one on the surface of the protein drastically reduces the solubility of the deoxygenated sickle hemoglobin (HbS) 1 , leading to its polymerization into long helical fibers that are responsible for the clinical manifestations of sickle cell disease. Electron microscopy and crystallographic studies have suggested that both the deoxy HbS crystal and fiber are constructed from the same "Wishner-Love" double strands (2-5). The model of the fiber structure derived from these analyses consists of seven Wishner-Love double strands (6 -9).The polymerization process is triggered by lateral interactions of the donor Val-6␤ of a tetramer of one strand of the double strand with the acceptor pocket at the EF corner (elicited mainly by ␤Phe-85 and ␤Leu-88) of the ␤-chain of an adjacent molecule present in the second strand of the double strand. Subsequent intra-double strand and inter-double strand interactions involving several amino acid residues from both ␣-and ␤-chains contribute to the stabilization of the fiber structure. The polymerization-impairing o...

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Mutational analysis of phenylalanine β85 in the valine β6 acceptor pocket during hemoglobin S polymerization

Cited by 11 publications

References 21 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

A Role for the α113 (GH1) Amino Acid Residue in the Polymerization of Sickle Hemoglobin

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