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

ABSTRACT: Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to ?-hemoglobin (?-Hb) or ?-globin and maintains it in a soluble state until its association with the ?-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of ?-Hb. To investigate the degree of interaction of the various regions of the ?-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the ?-globin H helix: five truncated ?-Hbs ?-Hb1-138, ?-Hb1-134, ?-Hb1-126, ?-Hb1-123, ?-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 ?-Hb was similar to that of the wild type ?-Hb except the shortest protein ?-Hb1-117 which displayed a decreased expression. While truncated GST-?-Hb1-138 and GST-?-Hb1-134 were normally soluble; the shorter globins GST-?-Hb1-126 and GST-?-Hb1-117 were obtained in very low quantities, and the truncated GST-?-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated ?-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 ?-globin for the heme. Upon addition of ?-Hb, the increase in fluorescence indicates the replacement of AHSP by ?-Hb. The CO binding kinetics of different truncated AHSPWT/?-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 ?-globin chain.

SUBMITTER: Domingues-Hamdi E 

PROVIDER: S-EPMC4219717 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature