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Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Domingues-Hamdi E, Vasseur C, Fournier JB, Marden MC, Wajcman H, Baudin-Creuza V - PLoS ONE (2014)

Bottom Line: SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression.The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition.The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.

ABSTRACT
Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

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Ligand binding studies of the various truncated GST-AHSPWT/GST-α-Hb complexes (A) and after addition of β-Hb in the presence of IHP (B).The CO recombination kinetics reveal whether the protein complexes display the known rates for the classical R and T states, or the reference intermediate rate for the AHSPWT/α-HbWT complex. The truncated forms of α-Hbs complexed with AHSP show less of the I state, indicating a modified interaction. Usually, the addition of β-Hb in the presence of IHP provokes a replacement of the AHSP by the β-Hb and leads to formation of Hb dimers and tetramers which display the usual slow T-state kinetics; however, the truncated forms displayed little of this allosteric form. Experimental conditions were 50 mM Bis-Tris buffer at pH 7.0, 100 mM CO at 25°C in absence or in presence of IHP at a final concentration of 1 mM. The concentration of different complexes on heme basis varies between 3.4 and 10.6 µM depending on the truncation.
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pone-0111395-g008: Ligand binding studies of the various truncated GST-AHSPWT/GST-α-Hb complexes (A) and after addition of β-Hb in the presence of IHP (B).The CO recombination kinetics reveal whether the protein complexes display the known rates for the classical R and T states, or the reference intermediate rate for the AHSPWT/α-HbWT complex. The truncated forms of α-Hbs complexed with AHSP show less of the I state, indicating a modified interaction. Usually, the addition of β-Hb in the presence of IHP provokes a replacement of the AHSP by the β-Hb and leads to formation of Hb dimers and tetramers which display the usual slow T-state kinetics; however, the truncated forms displayed little of this allosteric form. Experimental conditions were 50 mM Bis-Tris buffer at pH 7.0, 100 mM CO at 25°C in absence or in presence of IHP at a final concentration of 1 mM. The concentration of different complexes on heme basis varies between 3.4 and 10.6 µM depending on the truncation.

Mentions: Tetrameric Hb presents CO binding kinetics including a rapid phase (CO association rate of 6 µM−1s−1) and a slow phase (CO association rate of 0.2 µM−1s−1) corresponding to the R and T states, respectively. Unlike isolated α- and β-Hb subunits, which display CO binding kinetics typical of the R-state, α-Hb in the presence of AHSP exhibits a rate of about 2 µM−1s−1, intermediate to the R and T states of tetrameric Hb, and was thus denoted as the I phase [21]. We measured the influence of α-Hb chain truncation on CO recombination kinetic of different complexes (Figure 8A). These measurements were made directly on GST-AHSPWT/GST-α-Hb complexes because the GST moiety does not interfere with the CO recombination kinetics [19], [21]. Unlike AHSPWT/α-HbWT complex, the CO binding kinetic of different truncated complexes did not show mainly the intermediate phase but a rapid phase typical of R-state with a CO association rate of 6.5 µM−1s−1. The α-Hb1-138 complex exhibits nearly 100% of the rapid phase. The other truncated α-Hb complexes display between 10% and 20% of the I phase (Figure 8A and Table 4).


Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Domingues-Hamdi E, Vasseur C, Fournier JB, Marden MC, Wajcman H, Baudin-Creuza V - PLoS ONE (2014)

Ligand binding studies of the various truncated GST-AHSPWT/GST-α-Hb complexes (A) and after addition of β-Hb in the presence of IHP (B).The CO recombination kinetics reveal whether the protein complexes display the known rates for the classical R and T states, or the reference intermediate rate for the AHSPWT/α-HbWT complex. The truncated forms of α-Hbs complexed with AHSP show less of the I state, indicating a modified interaction. Usually, the addition of β-Hb in the presence of IHP provokes a replacement of the AHSP by the β-Hb and leads to formation of Hb dimers and tetramers which display the usual slow T-state kinetics; however, the truncated forms displayed little of this allosteric form. Experimental conditions were 50 mM Bis-Tris buffer at pH 7.0, 100 mM CO at 25°C in absence or in presence of IHP at a final concentration of 1 mM. The concentration of different complexes on heme basis varies between 3.4 and 10.6 µM depending on the truncation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219717&req=5

pone-0111395-g008: Ligand binding studies of the various truncated GST-AHSPWT/GST-α-Hb complexes (A) and after addition of β-Hb in the presence of IHP (B).The CO recombination kinetics reveal whether the protein complexes display the known rates for the classical R and T states, or the reference intermediate rate for the AHSPWT/α-HbWT complex. The truncated forms of α-Hbs complexed with AHSP show less of the I state, indicating a modified interaction. Usually, the addition of β-Hb in the presence of IHP provokes a replacement of the AHSP by the β-Hb and leads to formation of Hb dimers and tetramers which display the usual slow T-state kinetics; however, the truncated forms displayed little of this allosteric form. Experimental conditions were 50 mM Bis-Tris buffer at pH 7.0, 100 mM CO at 25°C in absence or in presence of IHP at a final concentration of 1 mM. The concentration of different complexes on heme basis varies between 3.4 and 10.6 µM depending on the truncation.
Mentions: Tetrameric Hb presents CO binding kinetics including a rapid phase (CO association rate of 6 µM−1s−1) and a slow phase (CO association rate of 0.2 µM−1s−1) corresponding to the R and T states, respectively. Unlike isolated α- and β-Hb subunits, which display CO binding kinetics typical of the R-state, α-Hb in the presence of AHSP exhibits a rate of about 2 µM−1s−1, intermediate to the R and T states of tetrameric Hb, and was thus denoted as the I phase [21]. We measured the influence of α-Hb chain truncation on CO recombination kinetic of different complexes (Figure 8A). These measurements were made directly on GST-AHSPWT/GST-α-Hb complexes because the GST moiety does not interfere with the CO recombination kinetics [19], [21]. Unlike AHSPWT/α-HbWT complex, the CO binding kinetic of different truncated complexes did not show mainly the intermediate phase but a rapid phase typical of R-state with a CO association rate of 6.5 µM−1s−1. The α-Hb1-138 complex exhibits nearly 100% of the rapid phase. The other truncated α-Hb complexes display between 10% and 20% of the I phase (Figure 8A and Table 4).

Bottom Line: SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression.The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition.The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.

ABSTRACT
Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

Show MeSH
Related in: MedlinePlus