Activation of the low oxygen affinity-inducing potential of the Asn108(β)→Lys mutation of Hb-Presbyterian on intramolecular αα-fumaryl cross-bridging

Belur N. Manjula, Ashok Malavalli, Muthuchidambaram Prabhakaran, Joel M. Friedman, A. Seetharama Acharya

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The Asn108β→Lys mutation in hemoglobin (Hb-Presbyterian mutation) endows a low O2 affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two α99 lysine residues also induces a low O2 affinity into HbA. We have now engineered an αα-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O2 affinity-inducing structural perturbations. Despite the presence of the additional ε-amino group of Lys108(β) within the central cavity, the ε-amino group of Lys99(αα) of deoxy Hb-Presbyterian retained high selectivity for αα-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The αα-fumaryl cross-linking of Hb-Presbyterian reduced its O2 affinity much more significantly than that observed with HbA, indicating a synergy between the two low O2 affinity-inducing structural perturbations. Apparently, the αα-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O2 affinity-inducing potential of Lys108(β) that is generally activated in the presence of chloride. The synergy between the Asn108(β)→Lys mutation and the αα-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, αα-fumaryl Hb-Presbyterian accessed a low O2 affinity T-state that is accessed by HbA, αα-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the αα-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(α) and Lys108(β) [presumably His103(α) and/or Glu101(β)] to compensate for the extra positive charge of Lys108(β). Molecular modeling studies identified two potential chloride binding sites per αβ dimer within the middle of the central cavity of αα-fumaryl HbA involving residues His103(α), Arg104(β) and Asn108(β). The affinity of these sites is increased in αα-fumaryl Hb-Presbyterian as a result of the Asn108(β)→Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the αα-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the αα-fumaryl-Hb Presbyterian to access a low O2 affinity T-state structure much more readily than either Hb-Presbyterian or αα-fumaryl HbA.

Original languageEnglish (US)
Pages (from-to)359-366
Number of pages8
JournalProtein Engineering
Volume14
Issue number5
StatePublished - 2001

Fingerprint

Chemical activation
Oxygen
Mutation
Chlorides
Molecular modeling
Hemoglobin
Binding sites
Dimers
Functional groups
Ionization
Electrostatics
Proteins
hemoglobin Presbyterian
Ions
Isoelectric Focusing
Static Electricity
Lysine
Hemoglobins
Binding Sites

Keywords

  • αα-fumaryl cross-linking
  • Chloride binding
  • Hb-Presbyterian
  • Low oxygen affinity

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Activation of the low oxygen affinity-inducing potential of the Asn108(β)→Lys mutation of Hb-Presbyterian on intramolecular αα-fumaryl cross-bridging. / Manjula, Belur N.; Malavalli, Ashok; Prabhakaran, Muthuchidambaram; Friedman, Joel M.; Acharya, A. Seetharama.

In: Protein Engineering, Vol. 14, No. 5, 2001, p. 359-366.

Research output: Contribution to journalArticle

Manjula, Belur N. ; Malavalli, Ashok ; Prabhakaran, Muthuchidambaram ; Friedman, Joel M. ; Acharya, A. Seetharama. / Activation of the low oxygen affinity-inducing potential of the Asn108(β)→Lys mutation of Hb-Presbyterian on intramolecular αα-fumaryl cross-bridging. In: Protein Engineering. 2001 ; Vol. 14, No. 5. pp. 359-366.
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abstract = "The Asn108β→Lys mutation in hemoglobin (Hb-Presbyterian mutation) endows a low O2 affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two α99 lysine residues also induces a low O2 affinity into HbA. We have now engineered an αα-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O2 affinity-inducing structural perturbations. Despite the presence of the additional ε-amino group of Lys108(β) within the central cavity, the ε-amino group of Lys99(αα) of deoxy Hb-Presbyterian retained high selectivity for αα-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The αα-fumaryl cross-linking of Hb-Presbyterian reduced its O2 affinity much more significantly than that observed with HbA, indicating a synergy between the two low O2 affinity-inducing structural perturbations. Apparently, the αα-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O2 affinity-inducing potential of Lys108(β) that is generally activated in the presence of chloride. The synergy between the Asn108(β)→Lys mutation and the αα-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, αα-fumaryl Hb-Presbyterian accessed a low O2 affinity T-state that is accessed by HbA, αα-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the αα-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(α) and Lys108(β) [presumably His103(α) and/or Glu101(β)] to compensate for the extra positive charge of Lys108(β). Molecular modeling studies identified two potential chloride binding sites per αβ dimer within the middle of the central cavity of αα-fumaryl HbA involving residues His103(α), Arg104(β) and Asn108(β). The affinity of these sites is increased in αα-fumaryl Hb-Presbyterian as a result of the Asn108(β)→Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the αα-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the αα-fumaryl-Hb Presbyterian to access a low O2 affinity T-state structure much more readily than either Hb-Presbyterian or αα-fumaryl HbA.",
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T1 - Activation of the low oxygen affinity-inducing potential of the Asn108(β)→Lys mutation of Hb-Presbyterian on intramolecular αα-fumaryl cross-bridging

AU - Manjula, Belur N.

AU - Malavalli, Ashok

AU - Prabhakaran, Muthuchidambaram

AU - Friedman, Joel M.

AU - Acharya, A. Seetharama

PY - 2001

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N2 - The Asn108β→Lys mutation in hemoglobin (Hb-Presbyterian mutation) endows a low O2 affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two α99 lysine residues also induces a low O2 affinity into HbA. We have now engineered an αα-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O2 affinity-inducing structural perturbations. Despite the presence of the additional ε-amino group of Lys108(β) within the central cavity, the ε-amino group of Lys99(αα) of deoxy Hb-Presbyterian retained high selectivity for αα-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The αα-fumaryl cross-linking of Hb-Presbyterian reduced its O2 affinity much more significantly than that observed with HbA, indicating a synergy between the two low O2 affinity-inducing structural perturbations. Apparently, the αα-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O2 affinity-inducing potential of Lys108(β) that is generally activated in the presence of chloride. The synergy between the Asn108(β)→Lys mutation and the αα-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, αα-fumaryl Hb-Presbyterian accessed a low O2 affinity T-state that is accessed by HbA, αα-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the αα-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(α) and Lys108(β) [presumably His103(α) and/or Glu101(β)] to compensate for the extra positive charge of Lys108(β). Molecular modeling studies identified two potential chloride binding sites per αβ dimer within the middle of the central cavity of αα-fumaryl HbA involving residues His103(α), Arg104(β) and Asn108(β). The affinity of these sites is increased in αα-fumaryl Hb-Presbyterian as a result of the Asn108(β)→Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the αα-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the αα-fumaryl-Hb Presbyterian to access a low O2 affinity T-state structure much more readily than either Hb-Presbyterian or αα-fumaryl HbA.

AB - The Asn108β→Lys mutation in hemoglobin (Hb-Presbyterian mutation) endows a low O2 affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two α99 lysine residues also induces a low O2 affinity into HbA. We have now engineered an αα-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O2 affinity-inducing structural perturbations. Despite the presence of the additional ε-amino group of Lys108(β) within the central cavity, the ε-amino group of Lys99(αα) of deoxy Hb-Presbyterian retained high selectivity for αα-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The αα-fumaryl cross-linking of Hb-Presbyterian reduced its O2 affinity much more significantly than that observed with HbA, indicating a synergy between the two low O2 affinity-inducing structural perturbations. Apparently, the αα-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O2 affinity-inducing potential of Lys108(β) that is generally activated in the presence of chloride. The synergy between the Asn108(β)→Lys mutation and the αα-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, αα-fumaryl Hb-Presbyterian accessed a low O2 affinity T-state that is accessed by HbA, αα-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the αα-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(α) and Lys108(β) [presumably His103(α) and/or Glu101(β)] to compensate for the extra positive charge of Lys108(β). Molecular modeling studies identified two potential chloride binding sites per αβ dimer within the middle of the central cavity of αα-fumaryl HbA involving residues His103(α), Arg104(β) and Asn108(β). The affinity of these sites is increased in αα-fumaryl Hb-Presbyterian as a result of the Asn108(β)→Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the αα-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the αα-fumaryl-Hb Presbyterian to access a low O2 affinity T-state structure much more readily than either Hb-Presbyterian or αα-fumaryl HbA.

KW - αα-fumaryl cross-linking

KW - Chloride binding

KW - Hb-Presbyterian

KW - Low oxygen affinity

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