title: Mechanism of Human Apohemoglobin Unfolding.
abstract: Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1interface is more resistant to dissociation than the α1β1interface.
authors.affiliation: Department of BioSciences and ‡Department of Chemistry, Rice University , Houston, Texas 77251, United States.
authors.forename: Premila P
authors.initials: PP
authors.lastname: Samuel
authors.affiliation: Department of BioSciences and ‡Department of Chemistry, Rice University , Houston, Texas 77251, United States.
authors.forename: William C
authors.initials: WC
authors.lastname: Ou
authors.affiliation: Department of BioSciences and ‡Department of Chemistry, Rice University , Houston, Texas 77251, United States.
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
authors.affiliation: Department of BioSciences and ‡Department of Chemistry, Rice University , Houston, Texas 77251, United States.
authors.forename: John S
authors.identifier: 0000-0002-0760-5403
authors.initials: JS
authors.lastname: Olson
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry*; D006418:Heme / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011489:Protein Denaturation; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001059:Apoproteins; D006454:Hemoglobins; D011994:Recombinant Proteins; C023478:apohemoglobin; D006418:Heme; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D019791:Guanidine; D005998:Glycine