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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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Effect of truncation on the solubility of various truncated α-Hbs.The induced E.coli BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.
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pone-0111395-g003: Effect of truncation on the solubility of various truncated α-Hbs.The induced E.coli BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.

Mentions: After solubilization, only the GST-α-Hb1-138 and GST-α-Hb1-134 were observed in the supernatant by SDS-PAGE analysis (Figure 3A) indicating their good solubility. These results were confirmed by Western blotting using anti-α globin antibodies (Figure 3B). To evaluate the amount of truncated GST-α-Hbs remaining in the pellet, the insoluble fraction was also analyzed by SDS-PAGE (Figure 3C). The GST-α-Hbs are clearly present but less abundant in the case of the truncated GST-α-Hb1-138, which was mostly present in the supernatant (Table 3). The same trend was observed by Western blotting analysis (Figure 3D), indicating that the truncated α-Hb1-126, α-Hb1-123 and α-Hb1-117 are expressed but are poorly soluble.


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)

Effect of truncation on the solubility of various truncated α-Hbs.The induced E.coli BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111395-g003: Effect of truncation on the solubility of various truncated α-Hbs.The induced E.coli BL21(DE3) cells containing different pGEX-α-AHSP plasmids were disrupted. After centrifugation, soluble fractions were analyzed by (A) SDS-PAGE (12%) and by (B) Western Blotting using anti-α globin antibodies. The insoluble fractions were analyzed by (C) SDS-PAGE (12%) and by (D) Western Blotting using anti-α globin antibodies. Page ruler™ prestained protein ladder (Fermentas Thermo Fisher Scientific) was in the first lane.
Mentions: After solubilization, only the GST-α-Hb1-138 and GST-α-Hb1-134 were observed in the supernatant by SDS-PAGE analysis (Figure 3A) indicating their good solubility. These results were confirmed by Western blotting using anti-α globin antibodies (Figure 3B). To evaluate the amount of truncated GST-α-Hbs remaining in the pellet, the insoluble fraction was also analyzed by SDS-PAGE (Figure 3C). The GST-α-Hbs are clearly present but less abundant in the case of the truncated GST-α-Hb1-138, which was mostly present in the supernatant (Table 3). The same trend was observed by Western blotting analysis (Figure 3D), indicating that the truncated α-Hb1-126, α-Hb1-123 and α-Hb1-117 are expressed but are poorly soluble.

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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