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

PLoS One. 2014 Nov 4;9(11):e111395. doi: 10.1371/journal.pone.0111395. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blood Proteins / chemistry
  • Blood Proteins / genetics
  • Blood Proteins / metabolism*
  • Carbon Monoxide / chemistry
  • Carbon Monoxide / metabolism
  • Circular Dichroism
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • Heme / chemistry
  • Heme / metabolism*
  • Hemoglobins / chemistry
  • Hemoglobins / metabolism*
  • Humans
  • Kinetics
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Spectrophotometry, Ultraviolet
  • alpha-Globins / chemistry
  • alpha-Globins / genetics
  • alpha-Globins / metabolism*

Substances

  • AHSP protein, human
  • Blood Proteins
  • Hemoglobins
  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • alpha-Globins
  • Heme
  • Carbon Monoxide
  • Glutathione Transferase

Grants and funding

This work was supported by the Institut National de la Santé et de la Recherche Médicale and the University Paris XI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.