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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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Fluorescence emission spectra of AHSPWT/α-Hb complexes before and after addition of β-Hb.(A). Fluorescence emission spectra of AHSPWT/α-HbWT complex; (B). Fluorescence emission spectra of AHSPWT/α-Hb1-138 complex; (C). Fluorescence emission spectra of AHSPWT/α-Hb1-134 complex; (D). Fluorescence emission spectra of AHSPWT/α-Hb1-126 complex. The fluorescence emission spectrum of AHSPWT is shown in the figure 7A (solid black line). The concentrations are around 3 µM (on a heme basis) in PBS. Each different emission spectrum of AHSPWT/truncated α-Hb complex is normalized with respect to the emission maxima obtained for normal complex. The dashed lines illustrate the fluorescence emission spectra of different AHSPWT/α-Hb complexes after addition of β-Hb chains (2/3 equivalent).
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pone-0111395-g007: Fluorescence emission spectra of AHSPWT/α-Hb complexes before and after addition of β-Hb.(A). Fluorescence emission spectra of AHSPWT/α-HbWT complex; (B). Fluorescence emission spectra of AHSPWT/α-Hb1-138 complex; (C). Fluorescence emission spectra of AHSPWT/α-Hb1-134 complex; (D). Fluorescence emission spectra of AHSPWT/α-Hb1-126 complex. The fluorescence emission spectrum of AHSPWT is shown in the figure 7A (solid black line). The concentrations are around 3 µM (on a heme basis) in PBS. Each different emission spectrum of AHSPWT/truncated α-Hb complex is normalized with respect to the emission maxima obtained for normal complex. The dashed lines illustrate the fluorescence emission spectra of different AHSPWT/α-Hb complexes after addition of β-Hb chains (2/3 equivalent).

Mentions: The fluorescence energy transfer technique was used to investigate the interaction between AHSP and truncated α-Hb within the AHSPWT/α-Hb complexes. AHSP has a single Trp (position 44) and exhibits a fluorescence spectrum typical of an exposed Trp (solide black line in Figure 7A). α-Hb has also a single Trp at position 14 (A2 in the A helix) but the α-Hb fluorescence is highly quenched by its heme group. It is important to note that there is no difference in fluorescence emission spectra between AHSPWT/native α-Hb and AHSPWT/α-HbWT complexes as illustrated in the Figure S1. In Figure 7 are illustrated the different emission spectra obtained for different complexes after normalization with respect to the emission maxima obtained with the WT complex. The emission spectrum of AHSPWT/α-HbWT complex is highly decreased compared to AHSPWT (solid red line and solid black line respectively, in Figure 7A), the quenching of AHSP fluorescence intensity demonstrating the interaction between AHSP and α-Hb [21]. Only the AHSPWT/α-Hb1-138 complex exhibits an emission spectrum with a slightly decreased fluorescence signal compared to that AHSPWT/α-HbWT complex (solid line in Figure 7B). The AHSPWT/α-Hb1-134 and AHSPWT/α-Hb1-126 complexes (solid lines in Figure 7C and 7D) have emission spectra with an increased amplitude, due to less heme molecule and therefore less quenching. These data are in agreement with absorbance spectra results (Figure 5). For all truncated complexes, one observes a red shift of fluorescence indicating that the environment of Trp is more polar.


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)

Fluorescence emission spectra of AHSPWT/α-Hb complexes before and after addition of β-Hb.(A). Fluorescence emission spectra of AHSPWT/α-HbWT complex; (B). Fluorescence emission spectra of AHSPWT/α-Hb1-138 complex; (C). Fluorescence emission spectra of AHSPWT/α-Hb1-134 complex; (D). Fluorescence emission spectra of AHSPWT/α-Hb1-126 complex. The fluorescence emission spectrum of AHSPWT is shown in the figure 7A (solid black line). The concentrations are around 3 µM (on a heme basis) in PBS. Each different emission spectrum of AHSPWT/truncated α-Hb complex is normalized with respect to the emission maxima obtained for normal complex. The dashed lines illustrate the fluorescence emission spectra of different AHSPWT/α-Hb complexes after addition of β-Hb chains (2/3 equivalent).
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pone-0111395-g007: Fluorescence emission spectra of AHSPWT/α-Hb complexes before and after addition of β-Hb.(A). Fluorescence emission spectra of AHSPWT/α-HbWT complex; (B). Fluorescence emission spectra of AHSPWT/α-Hb1-138 complex; (C). Fluorescence emission spectra of AHSPWT/α-Hb1-134 complex; (D). Fluorescence emission spectra of AHSPWT/α-Hb1-126 complex. The fluorescence emission spectrum of AHSPWT is shown in the figure 7A (solid black line). The concentrations are around 3 µM (on a heme basis) in PBS. Each different emission spectrum of AHSPWT/truncated α-Hb complex is normalized with respect to the emission maxima obtained for normal complex. The dashed lines illustrate the fluorescence emission spectra of different AHSPWT/α-Hb complexes after addition of β-Hb chains (2/3 equivalent).
Mentions: The fluorescence energy transfer technique was used to investigate the interaction between AHSP and truncated α-Hb within the AHSPWT/α-Hb complexes. AHSP has a single Trp (position 44) and exhibits a fluorescence spectrum typical of an exposed Trp (solide black line in Figure 7A). α-Hb has also a single Trp at position 14 (A2 in the A helix) but the α-Hb fluorescence is highly quenched by its heme group. It is important to note that there is no difference in fluorescence emission spectra between AHSPWT/native α-Hb and AHSPWT/α-HbWT complexes as illustrated in the Figure S1. In Figure 7 are illustrated the different emission spectra obtained for different complexes after normalization with respect to the emission maxima obtained with the WT complex. The emission spectrum of AHSPWT/α-HbWT complex is highly decreased compared to AHSPWT (solid red line and solid black line respectively, in Figure 7A), the quenching of AHSP fluorescence intensity demonstrating the interaction between AHSP and α-Hb [21]. Only the AHSPWT/α-Hb1-138 complex exhibits an emission spectrum with a slightly decreased fluorescence signal compared to that AHSPWT/α-HbWT complex (solid line in Figure 7B). The AHSPWT/α-Hb1-134 and AHSPWT/α-Hb1-126 complexes (solid lines in Figure 7C and 7D) have emission spectra with an increased amplitude, due to less heme molecule and therefore less quenching. These data are in agreement with absorbance spectra results (Figure 5). For all truncated complexes, one observes a red shift of fluorescence indicating that the environment of Trp is more polar.

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