Hemoglobin Rothschild (β37(C3)Trp → Arg): A high/low affinity hemoglobin mutant

In hemoglobin Rothschild arginine replaces the normal tryptophan at β37(C3), at α₁β₂ contact. Residue β37 is in close proximity to Argα92 (FG4). Substitution of Trp by Arg at β37 results in two positively charged Arg residues at FG4 and C3 facing each other, a situation that would destabilize the subunit constraints essential for the tetrameric integrity of the molecule and for the reduced ligand affinity of unliganded normal HB³ compared to isolated chains.Our studies show liganded HbR is extensively dissociated into dimers and has a high ligand affinity in phosphate buffer and a low ligand affinity in bis-Tris at alkaline pH. Kinetic studies indicate that in the T state HbR has a higher ligand affinity than HbA. This is explained by reduced subunit constraints in the T state and dissociation of the monoliganded species (Hb₄L) into dimers. Kinetic studies also show that R state Hb Rothschild has lower ligand affinity than R state HbA. These results are explained on the basis of extensive dissociation of R state Hb Rothschild into dimers and lower ligand affinity of dimers as compared to triliganded tetramers (α₂β₂(O₂)₃). Kinetic data indicate that the lower ligand affinity of dimers (Hb Rothschild) as compared to that of triliganded tetramers (HbA) is due to the increased ligand dissociation rates in the case of oxyhemoglobin and reduced ligand combination in the case of carboxyderivatives. Both the CO combination reaction time-course around 425 nm and the O₂ dissociation rates at 437.8 nm indicate the presence of large α,β-chain differences in Hb Rothschild.     

Hb Villaverde [β 89 (F5) Ser → Thr]: the structural modification of an intrasubunit contact is responsible for a high oxygen affinity

Hb Villaverde [β89 (F5) Ser → Thr], identified in a Spanish patient, is a new human hemoglobin variant, electrophoretically silent, responsible for a severe erythrocytosis. This abnormal hemoglobin displays a very high oxygen affinity and a markedly reduced cooperativity that is partly restored in the presence of IHP. Determination of the structural abnormality was achieved on a mixture of the normal and abnormal β-chains. After isolation of the abnormal tryptic peptide by RP-HPLC, its sequence was determined by mass spectrometry. The structural abnormality disturbs the intrasubunit interaction between helices F and H and, thus, may weaken the C-terminal bonds of the deoxy conformation and the heme contacts of several hydrophobic residues. Hb Villaverbe demonstrates that this intrasubunit contact between helices F and H is essential for the cohesion of the hemoglobin molecule.     

Hemoglobin Stanleyville II (α78 Asn→Lys) is associated with a 3.7-kb α-globin gene deletion

Summary A family with one homozygote and three heterozygotes for hemoglobin Stanleyville II (78 AsnLys) has been analyzed by -globin gene mapping. The pattern of restriction fragments with the enzymes BamHI and BglII demonstrated that the globin variant is associated with a 3.7-kb -globin gene deletion.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Hemoglobin roanne [α94(G1) asp å glu]: a variant of the α1β2 interface with an unexpected high oxygen affinity

In hemoglobin (Hb) Roanne, the aspartate residue α 94(G1) is replaced by a glutamic acid. This residue plays a key role in the structural changes affecting the α1β2 contact area during the deoxy- to oxy-state transition in the hemoglobin molecule. Aspartate α94(G1) is involved in several contacts both in the deoxy- and oxy-structures. The most important of those is a hydrogen bond with asparagine β102 (G4), stabilizing the oxygenated structure. Alteration of this contact usually leads to a decrease in oxygen affinity. Hb Roanne is the first example in which an increased oxygen affinity was found as a result of a structural modification at this position. Functional data suggested that the mechanisms responsible for this altered property are a destabilisation of the T-structure and a modification of the allosteric equilibrium.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Homoglobin Rouen (α-140 (HC2) Tyr→His): alteration of the α-chain C-terminal region and moderate increase in oxygen affinity

Hb Rouen (α140 (HC2) Tyr→His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the α-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the β-chain. This model provides additional evidence for the inequivalence between the α- and β-subunits.     

Hemoglobin calais [β 76 (E20) Ala → pro]: a hemoglobin variant with decreased intrinsic oxygen affinity

Hb Calais [β 76 (E20) Ala → Pro] is a new human hemoglobin variant displaying a decreased oxygen affinity. The only electrophoretical difference with Hb A was a slight more acidic isoelectric point. A 2-fold decrease in the oxygen affinity was found by equilibrium measurements performed in a suspension of intact red blood cells and in the lysate. It was confirmed by kinetic studies of the purified abnormal hemoglobin. The rte of methamoglobin formation at 37°C of Hb Calais was also increased realtive to Hb A. The mechanism by which the Pro for Ala substitution of an external residue in the β-chains results in these profound functional abnormalities is nuclear. Subtle changes at the heme pocket, at a distance from teh mutation, may be a plausible explanation for the effects observed.     

Synthesis and characterization of tetrairon-tungsten cluster complex containing a μ4, η-carbonyl ligand

Reaction of [(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (1) with (CO)₄W(CH₃CN)₂ at ambient temperature affords [(CO)₄W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (2) as the major product, together with a small amount of [(CO)₅W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (3). Compound 3 can be obtained in good yield by treating (CO)₅W(CH₃CN) with equal molar of 1, and reaction of 3 with Me₃NO in acetonitrile solvent produces 2 exclusively. The crystal structures of 2 and 3 have been determined by an X-ray diffraction study. Compound 2 contains an interesting μ₄, η²-CO ligand, where two electrons donated by the carbon atom are involved to bridge a Fe₃ face and two electrons from oxygen are donated to the tungsten(0) atom.     

Map2k4δ — Identification and functional characterization of a novel Map2k4 splice variant

Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.     

Hemoglobin tilburg: α2-ß2 73 (E 17) Asp→Gly: A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the ß-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp→Asn), Hb Mobile (Asp→Val) and Hb Vancouver (Asp→Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at ß 73.     

New examples of μ-η:η-disulfido dicopper(II,II) complexes with bis(tetramethylguanidine) ligands

The new ligand N,N′-(2-methyl-2-(2-pyridyl)propan-1,3-diyl)bis(tetramethylguanidine) (L) and its four-coordinate, distorted square-planar copper(II) complex [CuLCl₂] (1) were synthesized and structurally characterized. Similarly, bis(μ-OH)dicopper(II,II) complex [Cu₂L₂(OH)₂](OTf)₂ (2) was synthesized and structurally characterized. The pyridyl group in L does not coordinate in either 1 or 2. New examples of μ-η²:η²-disulfido dicopper(II,II) complexes were synthesized by treating a copper(I) complex of either L or L′ [L′ = 2′,2′-(propane-1,3-diyl)bis(1,1,3,3-tetramethylguanidine)] with elemental sulfur. [Cu₂L₂(S₂)](PF₆)₂ (3) and [Cu₂(S₂)](PF₆)₂ (4) were both structurally characterized, and both structures have two copper(II) ions bridged by a disulfido ligand in a μ-η²:η²-manner. The ligands L and L′ coordinate in a bidentate fashion (like 1 and 2, the pyridyl ring does not coordinate in 3), and the geometry around the copper ions in 3 and 4 is distorted square planar. The metrical parameters of 3 and 4 were found to be similar to other μ-η²:η²-disulfido dicopper(II,II) complexes, and the Cu-S and Cu···Cu distances are among the shortest reported for this class of copper disulfide dimers.     

Synthesis and characterisation of κ-P and κ-P,N palladium(II) complexes of the open cage water soluble aminophosphine PTN

New palladium(II) complexes containing the water soluble aminophosphine PTN ligand (PTN = 7-phospha-3,7-dimethyl-1,3,5-triazabicyclo[3.3.1]nonane) in 1:1 and 1:2 ratio Pd/PTN ligand, respectively, were prepared and fully characterised by mono and bidimensional ³¹P, ¹H and ¹³C NMR techniques showing that PTN can adopt both κ¹-P and κ²-P,N coordination modes. The complexes with Pd/PTN ratio 1:2 are highly soluble in water at room temperature. Suitable crystals for X-ray structure determination were obtained for the neutral complex κ²-P,N-Pd(PTN)(OAc)₂ (1) and for the monocationic complex [Pd(κ²-P,N-PTN)(κ¹-P-PTN)Cl][PF₆] (5).     

On the Activation of Integrin αIIbβ3: Outside-in and Inside-out Pathways

Integrin αIIbβ3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin αIIbβ3 is widely known to regulate the process of thrombosis via activation at its cytoplasmic side by talin and interaction with the soluble fibrinogen. It is also reported that three groups of interactions restrain integrin family members in the inactive state, including a set of salt bridges on the cytoplasmic side of the transmembrane domain of the integrin α- and β-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the α- and β-subunits that is known as the outer membrane clasp, and the key interaction group of the βA domain (located on the β-subunit head domain) with the βTD (proximal to the plasma membrane on the β-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the βTD and βA domains have remained ambiguous. In this study, we use molecular dynamics models to take a comprehensive outside-in and inside-out approach at exploring how integrin αIIbβ3 is activated. First, we show that talin’s interaction with the membrane-proximal and membrane-distal regions of integrin cytoplasmic-transmembrane domains significantly loosens the inner membrane clasp. Talin also interacts with an additional salt bridge (R734-E1006), which facilitates integrin activation through the separation of the integrin’s α- and β-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin αIIbβ3. Finally, we show that the interaction of the Arg of the RGD sequence may activate integrin via disrupting the key interaction group between K350 on the βA domain and S673/S674 on the βTD.     

Total synthesis of a cholinergic neuron-specific ganglioside GT1aα: A high affinity ligand for myelin-associated glycoprotein (MAG)

An efficient total synthesis of a cholinergic neuron-specific ganglioside GT1a (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4Cer) is described. The suitably protected sialyl-(26)-gangliotriose (III⁶NeuAc-GgOse3) derivative was glycosylated with the phenyl 2-thioglycoside of sialic acid in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) in acetonitrile medium, giving the disialogangliotriose (III⁶NeuAcII³NeuAc-GgOse3) derivative which contains both sialyl-(26)-GalNAc and sialyl-(23)-Gal structures (Route I). This pentasaccharide was efficiently synthesized also by the coupling of (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-2-deoxy-3,4--isopropylidene-2-phthalimido-D-galactopyranosyl trichloroacetimidate with 2-(trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-(2,6-di--benzyl--D-galactopyranosyl)-(14)-2,3,6-tri--benzyl--D-glucopyranoside, followed by conversion of the phthalimido group to the acetamido group (Route II). -Deisopropylidenation and further glycosylation with methyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-2,4,6-tri--benzoyl-1-thio--D-galactopyranoside, promoted by dimethyl(methylthio)sulfonium triflate (DMTST), gave the desired trisialogangliotetraose (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4) derivative, which was converted stepwise into the title ganglioside GT1a by the introduction of the ceramide part and then complete deprotection. The ganglioside obtained was shown to be identical with the native GT1a on TLC-immunostaining.     

Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia

We have analyzed the hemoglobins of a young German patient with β-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the β-globin through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the $\text{G}\text{T}\text{G}\text{→}\text{G}\text{G}\text{G}$ mutation at codon 126 leading to the synthesis of Hb Dhoburi or α₂β₂126(H₄)Val→Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G→C) mutation that leads to a rather severe β⁺-thalassemia and the $\text{G}\text{TG}\text{→}\text{A}\text{TG}$ mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered β-chain variant, named Hb Baden, was present for only 2–3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G→C) thalassemic mutation. The chromosome with the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) and the IVS-I-5 (G→C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G→C) mutation living in different countries failed to detect the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) change.     

Isolation and characterization of dinoflagellate and chrysophyte cytochrome-f (553–4)

Cytochrome-f (553–4) was isolated from mass cultures of the six dinoflagellates, Amphidinium carterae (2 strains), Cachonina niei, Glenodinium sp., Peridinium foliaceum, Gonyaulax polyedra, and one chrysophyte, Cricospherae carterae. Sonication of whole cell suspensions released the water-soluble protein, which was then purified by vacuum dialysis, salt fractionation and column chromatography. Reduced forms of isolated cytochromes had absorption maxima at 270–6, 316–8, 415–6, 522–3 and 553–4 rim. The α-absorption peak was asymmetrical. MW's, as determined by SDS polyacrylamide gel electrophoresis, ranged from 10 700 to 13 500. Amino acid analysis of C. niei cytochrome-f revealed 102 residues, with a composite MW of 10836. Purified cytochromes had isoelectric points ranging from 3·45 to 4·25 and oxidation-reduction potentials ranging from +0·374 to +0·351 V.     

Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα(4) with affinity for the volatile anesthetic halothane

A monomeric four-α-helix bundle protein Aα(4) was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B', A'. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A', B and B', respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI-TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα(4). Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα(4) is considerably more stable compared to the dimeric di-α-helical protein (Aα(2)-L38M)(2). The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα(4) bound halothane with a dissociation constant (K(d)) of 0.93±0.02mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.     

Altered Backbone and Side-Chain Interactions Result in Route Heterogeneity during the Folding of Interleukin-1β (IL-1β)

Deletion of the β-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1β into antagonist activity. Conversely, circular permutations of IL-1β conserve the functional folding route as well as the agonist activity. These two extremes in the folding-functional interplay beg the question of whether mutations in IL-1β would result in changes in the populations of heterogeneous folding routes and the signaling activity. A series of topologically equivalent water-mediated β-strand bridging interactions within the pseudosymmetric β-trefoil fold of IL-1β highlight the backbone water interactions that stabilize the secondary and tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape of IL-1β. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the population of folding routes accessed during folding and signaling activity.     

Hemoglobin J Cairo: β 65 (E9) Lys → Gln,a new hemoglobin variant discovered in an Egyptian family

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found.The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine β 65 is replaced by a glutamine. The new Hb mutant is designated hemoglobin J Cairo β 65 (E9) Lys → Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.