title: Protein conformational heterogeneity as a binding catalyst: ESI-MS study of hemoglobin H formation.
abstract: Our previous studies of hemoglobin tetramer assembly in vitro suggested that the initial step in the oligomerization process, which ultimately dictates the high fidelity of the heterotetramer (alpha*beta*)2 assembly, is the binding of a flexible heme-free beta-globin chain to a highly ordered heme-bound alpha*-globin. In this work, we extend these studies to investigate formation of the homotetrameric hemoglobin H, whose formation in vivo is a well-documented clinical consequence of significant overexpression of beta-globin in alpha-thalassemic disorders. Upon reconstitution of the isolated beta-globin with excess heme, the predominant species in the ESI mass spectrum corresponds to the homotetramer beta*4, alongside homodimeric species and monomeric beta-globin chains in both apo and holo forms. The assembly process of the hemoglobin H homotetramer apparently follows a scenario similar to that of a normal heterodimeric hemoglobin (alpha*beta*)2 species, with the asymmetric binding event between compact and flexible polypeptide chains being the initial step. The extreme importance of large-scale chain dynamics and conformational heterogeneity for the protein assembly process is highlighted by the inability of highly structured alpha-globins to undergo ordered oligomerization to form dimers and tetramers as opposed to indiscriminate aggregation.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
authors.forename: Wendell P
authors.initials: WP
authors.lastname: Griffith
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002417:Cattle; D005914:Globins / Q000737:chemistry; D006447:Hemoglobin H / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001059:Apoproteins; D012996:Solutions; D005914:Globins; D006447:Hemoglobin H