Dynamic properties of some β-chain mutant hemoglobins

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some β-chain mutant hemoglobins is studied in the temperature range 300–10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the overall bandwidth are singled out With this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wildtype and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme–CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the “polar isosteric” βVal-67(Ell) →Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue βLys-144(HC1) at the C-terminal and residue βCys-112(G14) at the α₁β₁ interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the β-N-terminal and at the α₁β₂ interface have no effect on the dynamic properties of the heme pocket. © 1995 Wiley-Liss, Inc.     

Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

The relative amounts of α- and β-globin mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (Hb F: α₂γ₂). Molecular hybridization assays used as probes full-length DNA copies of human α- and β-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of α-globin mRNA and gene DNA, but absence of β-globin mRNA and absence of β-globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the β-globin structural gene.     

Amino Acid Sequence of Kalinowaski's Tinamou (Nothoprocta Kalinowskii) Hemoglobin and the Rate of Evolution of Bird αD-Globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou α^D-, α^A-, and β-globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the α^D-globin chain. This has not been reported in the literature, except in pigeon embryonic α^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, α^D = α^A > β, was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me₂SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of $\text{α}\text{β}\text{-}\text{mRNA}$ is maintained during induction. In maximally induced cells, the $\text{α}\text{β}\text{-}\text{globin}$ mRNA ratios are approximately 1 in cells induced by Me₂SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.     

Expression of embryonic hemoglobin genes in mice heterozygous for α-thalassemia or β-duplication traits and in mice heterozygous for both traits

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous α-thalassemic embryos, expression of EI (x₂y₂) and EII (α₂y₂) is deficient because the x- and α-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- and α-globin genes indicates that these genes must be closely linked. Reduced x- and α-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y₄ hemoglobin also appears in β-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and β-globin genes. In double heterozygotes, which have a single functional allele of x- and α-globin genes and three functional alleles of y- and β-globin genes, synthesis of α and non-α chains is severely imbalanced and half of the total hemoglobin is y₄. Mouse y₄ has a high affinity for oxygen, P₅₀ of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be due in large part to oxygen deprivation to the tissues.     

The binding of 2,3-diphosphoglycerate as a conformational probe of human hemoglobins

Since 2,3-diphosphoglyeerate preferentially binds to deoxygenated hemoglobin A, this binding reaction can be used to detect the change in quaternary conformation of hemoglobin associated with the change in ligand state of the hemes. We have studied the binding to two M hemoglobins (M_HydePark, M_Milwaukee-1) that have the substituted chains in the ferric state, as well as to the mixed liganded hybrids α1₂β² and α²β1₂ (1 heme in cyanmet form) prepared from hemoglobins A and H. The studies demonstrate that when these hemoglobin variants and derivatives are deoxygenated, they bind the organic phosphate to an extent similar, but not identical, to that for fully deoxygenated hemoglobin A. The results indicate that removal of ligand from only two of the four hemes results in a change in quaternary structure to a deoxy-like conformation.     

Selective binding of antimicrobial porphyrins to the heme‐receptor IsdH‐NEAT3 of Staphylococcus aureus

The Isd (iron‐regulated surface determinant) system of the human pathogen Staphylococcus aureus is responsible for the acquisition of heme from the host organism. We recently reported that the extracellular heme receptor IsdH‐NEAT3 captures and transfers noniron antimicrobial porphyrins containing metals in oxidation state (III). However, it is unclear if geometric factors such as the size of the metal (ionic radius) affect binding and transfer of metalloporphyrins. We carried out an ample structural, functional, and thermodynamic analysis of the binding properties of antimicrobial indium(III)‐porphyrin, which bears a much larger metal ion than the iron(III) of the natural ligand heme. The results demonstrate that the NEAT3 receptor recognizes the In(III)‐containing PPIX in a manner very similar to that of heme. Site‐directed mutagenesis identifies Tyr642 as the central element in the recognition mechanism as suggested from the crystal structures. Importantly, the NEAT3 receptor possesses the remarkable ability to capture dimers of metalloporphyrin. Molecular dynamics simulations reveal that IsdH‐NEAT3 does not require conformational changes, or large rearrangements of the residues within its binding site, to accommodate the much larger (heme)₂ ligand. We discuss the implications of these findings for the design of potent inhibitors against this family of key receptors of S. aureus.     

Linkage of adult α- and β-globin genes in X. laevis and gene duplication by tetraploidization

We have used cloned adult X. laevis α- and β-globin cDNAs to analyze globin genes in X. laevis DNA. We detected α¹- and β¹-globin genes which contain intervening sequences and code for the major adult globins, plus additional diverged α²- and β²-globin genes of unknown coding potential. Unlike the case in mammals, the X. laevis α¹- and β¹-globin genes are closely linked and occur in the sequence 5′-α¹-9 kb-β¹-3′. The α²- and β²-globin genes are also linked, and analysis of globin genes in X. tropicalis suggests that this duplication of an α-β-globin gene pair in X. laevis is the result of chromosome duplication by tetraploidization. The close linkage of α- and β-globin genes in Xenopus provides evidence that vertebrate α- and β-globin genes evolved by tandem duplication of a single primordial globin gene.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.     

Soret Spectroscopic and Molecular Graphic Analysis of Human Semi-β-Hemoglobin Formation

The interaction of heme-free α (α^o) and heme-containing β (β^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^?1 s^?1. This was 100-fold more rapid than the reported rate of combination of α^h and β^h proteins. This encounter-like rate of semi-β-hemoglobin (α^oβ^h) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled α^o protein superimposed over native α^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the α₁β₁ interface, was apparently responsible for the enhanced association properties of the α^o protein.     

一个α,β地中海贫血复合家系β珠蛋白基因的进一步研究

 前文~[1]曾报道广西一个α,β地中海贫血复合家系的血红蛋白组成及α珠蛋白基因分析结果,并讨论了各成员可能的β珠蛋白基因结构情况。本文利用先进的PCR即基因扩增技术,结合特异寡核苷酸探针斑点杂交及扩增后直接测定DNA序列的技术,进一步研究并彻底搞请了该家系各成员的β珠蛋白基因结构情况。结果显示:母亲及两个弟弟都是编码子41—42TTCT四个碱基缺失造成框架位移所致β地中海贫血的杂合子。父亲与先证者的β基因均属正常。前三个成员均为α地贫复合β地贫,其α与β珠蛋白链合成的不均衡状态得到改善,贫血症状也明显轻。     

Human and rodent amyloid-β peptides differentially bind heme: Relevance to the human susceptibility to Alzheimer’s disease

Amyloid-β (Aβ) peptides are implicated in the neurodegeneration of Alzheimer’s disease (AD). We previously investigated the mechanism of neurotoxicity of Aβ and found that human Aβ (huAβ) binds and depletes heme, forming an Aβ-heme complex with peroxidase activity. Rodent Aβ (roAβ) is identical to huAβ, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAβ and huAβ. Unlike roAβ, huAβ binds heme tightly (K_d = 140 ± 60 nM) and forms a peroxidase. The plot of bound (huAβ-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAβ possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAβ (K_d = 210 ± 80nM). The plot of (roAβ-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAβ for heme (K_d = 1 μM). The effect of heme-binding to site-H on heme-binding to site-L in roAβ and huAβ is discussed. While both roAβ and huAβ form aggregates equally, rodents lack AD-like neuropathology. High huAβ/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Aβ-heme peroxidase contribute to huAβ’s neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Aβ explain tight heme-binding to huAβ and likely contribute to the increased human susceptibility to AD.     

The Primary Structure of β<Superscript>I</Superscript>-Chain of Hemoglobin from Snake Sindhi Krait (<Emphasis Type="Italic">Bungarus sindanus sindanus</Emphasis>)

The amino acid sequence of β^I-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of β^II, β^I, α^D and α^A-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of β^I globin chain from Sindhi Krait showed closest relationship with β^I globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of β^I globin chain of Sindhi Krait with human β chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αβ contact points and in regions directly not important for function.     

Studies on cobalt myoglobins and hemoglobins: XI. The interaction of carbon monoxide and oxygen with α and β subunits in iron-cobalt hybrid hemoglobins

An artificial hybrid hemoglobin, α(Co)₂β(Fe)₂, the α and β subunits of which carry cobaltous protoporphyrin IX and ferrous protoporphyrin IX, respectively, and its complementary hybrid α(Fe)₂β(Co)₂ were prepared and the properties of their ferrous subunits were examined by equilibrium and kinetic measurements with carbon monoxide as a ligand.The β(Fe)₂ subunits in fully deoxy α(Co)₂β(Fe)₂ exhibited a higher affinity for carbon monoxide than did the α(Fe)₂ subunits in fully deoxy α(Fe)₂β(Co)₂. Addition of 2 mm-inositol hexaphosphate decreased fourfold the carbon monoxide affinity of the α(Fe)₂ subunits in α(Fe)₂β(Co)₂ and by more than tenfold that of the β(Fe)₂ subunits in α(Co)₂β(Fe)₂ at pH 7.0. The higher affinity for carbon monoxide of the β(Fe)₂ subunits inα(Co)₂β(Fe)₂ than that of the α(Fe)₂ subunits in α(Fe)₂β(Co)₂ was caused by a smaller dissociation rate of the β(Fe)₂-carbon monoxide complex. These results are considered to underlie the different affinity for carbon monoxide of the α and β subunits in deoxy hemoglobin.Oxygen equilibria of the cobaltous subunits in iron-cobalt hybrid hemoglobins were also measured in the presence of carbon monoxide. The α(Co)₂ subunits in α(Co)₂β(FeCO)₂ showed a higher oxygen affinity than the β(Co)₂ subunits in α(FeCO)₂β(Co)₂. Inositol hexaphosphate lowered the oxygen affinity of the β(Co)₂ subunits in α(FeCO)₂β(Co)₂ by eight-fold, but that of the α(Co)₂ subunits in α(Co)₂β(FeCO)₂¦by only 1.6-fold. The magnitude of the alkaline Bohr effect, as defined by Δlog $\text{P}{}_{\mathrm{<mtext>m</mtext>}}\text{Δ}\text{pH}$ was found to be ?0.34 and ?0.14 for α(FeCO)₂β(Co)₂ and α(Co)₂β(FeCO)₂, respectively, in 0.1 m-phosphate buffer at 15 °C.The rate of oxygenation and deoxygenation of the cobaltous subunits in iron-cobalt hybrid hemoglobins in the presence of carbon monoxide was determined by a temperature-jump relaxation method in 0.1 m-phosphate buffer with and without inositol hexaphosphate. Their relaxation spectra were of a single exponential character and differed from that of cobalt hemoglobin. Without inositol hexaphosphate, the association rate constants for both α(Co)₂β(FeCO)₂ and α(FeCO)₂β(Co)₂ were close to that for cobalt hemoglobin, whereas the dissociation rate constants for iron-cobalt hybrid hemoglobins were smaller than that for cobalt hemoglobin by more than fourfold. Inositol hexaphosphate affected both the association and dissociation rates of α(FeCO)₂β(Co)₂ but did so to a lesser extent than those of α(Co)₂β(FeCO)₂.These observations suggest strongly a different role for the α and β subunits in the co-operative oxygenation and alkaline Bohr effect of hemoglobin.     

Localization of integrin heterodimer α9β1 on the surface of uterine endometrial stromal and epithelial cells in mice

ABSTRACT

Previously, we reported that endometrial stromal (ES) and endometrial epithelial (EE) cells did not attach to tenascin C, indicating the absence of active integrin α₉β₁ on the surface of mouse ES and EE cells. However, that study used recombinant tenascin C without fibronectin (FN) type III repeats interacting with integrin heterodimers. Therefore, we re-evaluated the presence of integrin α₉β₁ actively functioning on the surface of mouse ES and EE cells using full-length native tenascin C with FN type III repeats. The functionality of integrin α₉β₁ was confirmed using attachment and antibody inhibition assays. Both mouse ES and EE cells showed significantly increased adhesion to native tenascin C, and functional blocking of integrin α₉β₁ significantly inhibited adhesion to native tenascin C. These results demonstrate that the integrin α₉ and β₁ subunits function as active heterodimers on the plasma membrane of mouse ES and EE cells, respectively.     

Air-stable,heme-like water-soluble iron(II) porphyrin: in situ preparation and characterization

Preparation of the water-soluble, kinetically labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)porphyrin, Fe(II)TPPS⁴⁻, has been realized in neutral or weakly acidic solutions containing acetate buffer. The buffer played a double role in these systems: it was used for both adjusting pH and, via formation of an acetato complex, trapping trace amounts of iron(III) ions, which would convert the iron(II) porphyrins to the corresponding iron(III) species. Fe(II)TPPS⁴⁻ proved to be stable in these solutions even after saturation with air or oxygen. In the absence of acetate ions, however, iron(II) ions play a catalytic role in the formation of iron(III) porphyrins. While the kinetically inert iron(III) porphyrin, Fe(III)TPPS³⁻, is a regular one with no emission and photoredox properties, the corresponding iron(II) porphyrin displays photoinduced features which are typical of sitting-atop complexes (redshifted Soret absorption and blueshifted emission and Q absorption bands, photoinduced porphyrin ligand-to-metal charge transfer, LMCT, reaction). In the photolysis of Fe(II)TPPS⁴⁻ the LMCT process is followed by detachment of the reduced metal center and an irreversible ring-opening of the porphyrin ligand, resulting in the degradation of the complex. Possible oxygen-binding ability of Fe(II)TPPS⁴⁻ (as a heme model) has been studied as well. Density functional theory calculations revealed that in solutions with high acetate concentration there is very little chance for iron(II) porpyrin to bind and release O₂, deviating from heme in a hydrophobic microenvironment in hemoglobin. In the presence of an iron(III)-trapping additive that is much less strongly coordinated to the iron(II) center than the acetate ion, Fe(II)TPPS⁴⁻ may function as a heme model.     

Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co²⁺-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO₂). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.     

Dissecting intrinsic and ligand-induced structural communication in the β3 headpiece of integrins

BackgroundGraph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes.MethodsProtein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins α_Vβ₃ and α_IIbβ₃ in their apo and ligand-bound states.ResultsClosed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states.ConclusionsAllosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process.General significanceThe study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.