title: Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.
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.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.
authors.forename: Elisa
authors.initials: E
authors.lastname: Domingues-Hamdi
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Vasseur
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Fournier
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Marden
authors.affiliation: INSERM U955, Université Paris Est Créteil (UPEC), Hôpital Henri Mondor, Paris, France.
authors.forename: Henri
authors.initials: H
authors.lastname: Wajcman
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Baudin-Creuza
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D055542:alpha-Globins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D006454:Hemoglobins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D055542:alpha-Globins; D006418:Heme; D002248:Carbon Monoxide; D005982:Glutathione Transferase