title: Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y).
abstract: Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.
authors.affiliation: INSERM U.91, CNRS UA607, Hôpital Henri Mondor, Créteil, France.
authors.forename: C T
authors.initials: CT
authors.lastname: Craescu
authors.forename: J
authors.initials: J
authors.lastname: Mispelter
authors.forename: Y
authors.initials: Y
authors.lastname: Blouquit
mesh_terms: D006454:Hemoglobins / Q000235:genetics; D006455:Hemoglobins, Abnormal; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D011487:Protein Conformation
chemical_list: D006454:Hemoglobins; D006455:Hemoglobins, Abnormal