ABSTRACT
The liganded (R-state) form of sickle cell haemoglobin (HbS) is of particular relevance at non-polymerizing concentrations as oxy HbS exhibits unusual properties compared with oxy HbA: mechanical precipitability (resulting from surface denaturation), greater unfolding at an air-water interface and a tendency to oxidize more readily. In human haemoglobins, the beta7 (A4) Glu residue forms an intrachain salt bridge with beta132 (H10) Lys in both liganded and deoxy structures. In the present study, recombinant haemoglobins with substitutions in the beta7 and beta132 sites were studied in order to determine the role of the beta7-beta132 salt bridge on Hb conformational integrity and stability. The elimination of this interhelix bridge correlates with enhanced surface denaturation and conformational alterations in the central cavity 2,3-diphosphoglycerate (DPG) cleft and alpha1beta2 interface. The A-helix beta7 Ala substitution generates a class of conformational change at the DPG pocket and alpha1beta2 interface that is distinct from that dictated by the H-helix beta132 Ala substitution. These results are significant with regard to the communication pathway between the alpha1beta1 and alpha1beta2 interfaces, and the new understanding of Hb allostery dependent upon tertiary structural constraints caused by effector binding to the R-state.
Subject(s)
Hemoglobins, Abnormal/genetics , Fluorometry , Hemoglobin A , Hemoglobin, Sickle , Humans , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Recombinant Proteins/geneticsABSTRACT
We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.
Subject(s)
Hemoglobins/chemistry , Amino Acid Substitution , Chromatography, Gel , Glutathione/metabolism , Hemoglobins/genetics , Hemoglobins/metabolism , Hot Temperature , Humans , Kinetics , Mass Spectrometry , Time FactorsABSTRACT
The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.
Subject(s)
Hemoglobin, Sickle/chemistry , Hemoglobin, Sickle/genetics , Dimerization , Glutamic Acid/chemistry , Humans , Hydrogen-Ion Concentration , Lysine/chemistry , Models, Molecular , Mutation , Point Mutation , Protein Conformation , Recombinant Proteins/chemistry , Spectrophotometry , Spectrum Analysis, Raman/methods , Ultraviolet RaysABSTRACT
Human and bovine hemoglobins (Hbs) exhibit several functional differences. They have a similar oxygen affinity in the presence of 2,3-diphosphoglycerate (2,3-DPG); however, bovine Hb has a greatly diminished 2,3-DPG effect, which itself is chloride dependent. The question is to determine whether these differences have a common structural origin, or whether they evolved in an independent fashion. The decreased 2,3-DPG effect can be partially reproduced by mutations at the effector binding sites, substituting the betaNA1 valine-NA2 histidine present in human Hb with a methionine. While changes of human Hb at these sites could provoke the bovine characteristic of the lower 2,3-DPG effect, the oxygen affinities of these mutated Hbs were not as low as that of the bovine Hb. Modifications responsible for tertiary structural modifications of helix A in human Hb might help shift the N-terminal methionine position, thereby locking helix A in place. We replaced the residues proline beta5(A2), arginine beta104(G6), and tyrosine beta130(H8) of human Hb by the residues present in bovine beta-globin, namely alanine, lysine, and phenylalanine, respectively. These mutations did not allow us to obtain a low oxygen affinity recombinant Hb (rHb). This indicates that other factors also influence oxygen binding and the effects are only partially coupled.
Subject(s)
Hemoglobins/chemistry , Hemoglobins/metabolism , Oxygen/metabolism , Adult , Amino Acid Substitution/genetics , Amino Acid Substitution/physiology , Animals , Binding Sites/genetics , Binding Sites/physiology , Cattle , Dimerization , Electrophoresis, Cellulose Acetate/methods , Hemoglobins/genetics , Hot Temperature/adverse effects , Humans , Mutagenesis, Site-Directed/genetics , Mutagenesis, Site-Directed/physiology , Oxygen/chemistry , Protein Binding/genetics , Protein Binding/physiology , Protein Denaturation , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The effects of the mutation beta9(A6)Ser --> Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the beta9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher-molecular-weight oligomeric fraction (30%). Reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ESI-MS) under denaturing conditions showed that the tetrameric fraction contained only normal alpha- and beta-chains, whereas the oligomeric fraction contained only normal alpha-chain and disulfide-linked beta(PA) dimer. Under native conditions, ESI-MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 A diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA-CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer-dimer allosteric interface. None of the other variants investigated formed such a large oligomer.
Subject(s)
Hemoglobins, Abnormal/chemistry , Chromatography, Gel , Cysteine/chemistry , Dimerization , Disulfides , Humans , Kinetics , Microscopy, Electron , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Protein Denaturation , Protein Structure, Quaternary , Protein Structure, Secondary , Serine/chemistry , Spectrometry, Mass, Electrospray Ionization , Time FactorsABSTRACT
Se ha descubierto en los Andes Peruanos un grupo de gallinas de altura (Gallus gallus) con alta afinidad de la hemoglobina por el oxígeno. Hemos estudiado el posible mecanismo molecular subyacente de esta adaptación a la altura. Se ha postulado pequeños cambios en la concentración intracelular de inositol pentafosfato (IPP), el principal efecto alostérico de la hemoglobina en los eritrocitos de aves. Hemos estudiado la sangre de gallinas andinas y de nivel del mar. Las afinidades de suspensiones frescas de eritrocitos están significativamente incrementadas en gallinas andinas comparadas con las de nivel del mar. Los valores de los coeficientes de Hill a saturación 50 por ciento (n50) son mayores para suspensiones de gallinas que para las de mamíferos. Esto podría sugerir la existencia de un proceso de agregación molecular dentro de las células deoxigenadas, altamente concentradas de ambos tipos de aves. Para las soluciones "desnudas" de hemolizados de ambos tipos de gallinas, las afinidades son idénticas en el buffer libre de fosfato, indicando que las afinidades intrínsecas de las Hbs de gallinas andinas y de nivel del mar son las mismas. Con la adición de inositol hexafosfato (IHP), un fuerte efecto alostérico de hemoglobinas de aves, observamos un incremento pequeño pero significativo (aproximadamente 20 por ciento) en la afinidad de los hemolisados de altura en relación a los de nivel del mar. Nuestros resultados también sugieren que la diferencia en la afinidad entre sangres de altura y de nivel del mar puede deberse a un proceso adaptativo posiblemente relacionado con una ligera disminución en la concentración y/o actividad del principal efector celular IPP, más bien que un anormalidad estructural de la hemoglobina.
