Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β108Asn → Lys)

David S. Gottfried, Belur N. Manjula, Ashok Malavalli, A. Seetharama Acharya, Joel M. Friedman

Research output: Contribution to journalArticle

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Abstract

HbPresbyterian (β108Asn → Lys, HbP) contains an additional positive charge (per αβ dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the ββ cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the β-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2,3- diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl- imidazole pathway linked to the His residues of the ββ cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the ββ cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

Original languageEnglish (US)
Pages (from-to)11307-11315
Number of pages9
JournalBiochemistry
Volume38
Issue number35
DOIs
StatePublished - Aug 31 1999

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Carbon Monoxide
Binding Sites
2,3-Diphosphoglycerate
Mutant Proteins
Dimers
Conformations
Chlorides
Proteins
Photons
Fluorescence
Ligation
Oxygen
Ligands
4-nitrophenyl acetate
imidazole

ASJC Scopus subject areas

  • Biochemistry

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Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β108Asn → Lys). / Gottfried, David S.; Manjula, Belur N.; Malavalli, Ashok; Acharya, A. Seetharama; Friedman, Joel M.

In: Biochemistry, Vol. 38, No. 35, 31.08.1999, p. 11307-11315.

Research output: Contribution to journalArticle

Gottfried, DS, Manjula, BN, Malavalli, A, Acharya, AS & Friedman, JM 1999, 'Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β108Asn → Lys)', Biochemistry, vol. 38, no. 35, pp. 11307-11315. https://doi.org/10.1021/bi9827464
Gottfried, David S. ; Manjula, Belur N. ; Malavalli, Ashok ; Acharya, A. Seetharama ; Friedman, Joel M. / Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β108Asn → Lys). In: Biochemistry. 1999 ; Vol. 38, No. 35. pp. 11307-11315.
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abstract = "HbPresbyterian (β108Asn → Lys, HbP) contains an additional positive charge (per αβ dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the ββ cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the β-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2,3- diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl- imidazole pathway linked to the His residues of the ββ cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the ββ cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.",
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T1 - Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (β108Asn → Lys)

AU - Gottfried, David S.

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AU - Malavalli, Ashok

AU - Acharya, A. Seetharama

AU - Friedman, Joel M.

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N2 - HbPresbyterian (β108Asn → Lys, HbP) contains an additional positive charge (per αβ dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the ββ cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the β-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2,3- diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl- imidazole pathway linked to the His residues of the ββ cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the ββ cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

AB - HbPresbyterian (β108Asn → Lys, HbP) contains an additional positive charge (per αβ dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the ββ cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the β-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2,3- diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl- imidazole pathway linked to the His residues of the ββ cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the ββ cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

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