Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala: isolation and functional properties of the heme saturated form.

Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala is a neutrally substituted unstable hemoglobin, exhibiting the same gross features as hemoglobin K?ln beta98 (FG 5) Val leads to Met. In addition to the presence of a deheminized fraction, a heme saturated abnormal hemoglobin was visualized and isolated by high resolution electrofocusing. By functional studies of the fully heminized form, a slightly increased oxygen affinity, an impairment of heme-heme interaction and a decreased response to organic phosphates were demonstrated. These functional perturbations point out the importance of the beta98 invariant valyl residue, in the quaternary contacts. They can account for the poor oxygen delivery of erythrocytes.     

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.     

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O?/NO chemistry, O? delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein.     

Hemoglobin Abruzzo (beta143 (H21) His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Abruzzo is an abnormal human hemoglobin with a substitution at a residue known to be involved in the binding of 2,3-diphosphoglyceric acid. It has increased oxygen affinity and reduced heme-heme interaction in the absence of organic or inorganic phosphate cofactors. In inorganic phosphate buffers the Bohr effect and heme-heme interaction are normal, but the oxygen affinity remains higher than that of hemoglobin A. CO combination in inorganic phosphate is more strongly autocatalytic than in normal hemoglobin and a slower rate of oxygen dissociation is observed. Although many of the functional differences of this variant may be attributed to the high oxygen affinity of the mutant beta chains, the interactions between subunits are also affected by the histidine to arginine substitution at beta143. Stripped hemoglobin Abruzzo appears to be significantly more dissociated than hemoglobin A. Kinetic studies indicate that interaction with organic or inorganic phosphates decreases its subunit dissociation. In all of the functional properties examined, hemoglobin Abruzzo is more sensitive to the allosteric influence of organic and inorganic anions than is hemoglobin A.     

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.     

Hb F-La Grange or alpha 2 gamma 2 101(G3)Glu----Lys; 75Ile; 136Gly: a high oxygen affinity fetal hemoglobin variant observed in a Caucasian newborn

The identification of a newly discovered gamma-chain variant is reported. This abnormal fetal hemoglobin is characterized by a Glu----Lys substitution at position 101(G3) of its gamma chain and was observed in a Caucasian baby girl. Because glutamic acid residue in position gamma 101 is involved in the alpha 1-gamma 2 chain contact, its replacement by a lysine residue results in changes in physicochemical and functional properties. The variant readily forms hybrid hemoglobins at room temperature, is mildly unstable at higher temperature, and has an increased oxygen affinity with a somewhat lower heme-heme interaction.     

Hemoglobin Cochin-Port-Royal: consequences of the replacement of the beta chain C-terminal by an arginine.

Hemoglobin Cochin Port-Royal beta 146 (HC3) His yields Arg is the second example in which the beta C-terminal residue is replaced. Owing to the known importance of His beta 146 in the co-operative effects of hemoglobin, the functional properties of this variant were carefully studied. It had a normal Hill coefficient but a reduced alkaline Bohr effect. However, the reduction in Bohr effect is less than the halving predicted from previous mutants and modified hemoglobins.     

NMR study of the sites of human hemoglobin acetylated by aspirin.

Acetylation of hemoglobin by aspirin and other acetylating agents has been used to generate hemoglobin analogs with altered structural and functional properties, and may prove useful in the treatment of sickle cell disease. We have studied the acetylation of human hemoglobin using [1'-(13)C]acetylsalicylic acid in combination with two-dimensional HMQC and HSQC NMR analysis. The spectra of the acetylated hemoglobin exhibit a number of well resolved resonances. Several spectral assignment strategies were used: blocking the 2, 3-DPG binding site non-covalently with inositol hexaphosphate or covalently with a cross-linking agent, selective carbamylation of the N-terminal valine amino groups with cyanate, spin-labeling the hemoglobin at betaCys93, and analysis of a hemoglobin triple mutant: betaV1MH2DeltaK144R, in which betaLys144 is replaced by an arginine residue. These studies support the conclusion that the most rapidly acetylated residue is betaLys82 rather than betaLys144, as previously reported. Further, it is apparent that acetyl betaLys82 can give rise to several resonances due to additional acetylation of betaLys82' or other nearby residues. An additional assignment strategy involving comparison of the chemical shifts of the acetyl resonances observed for adducts of diamagnetic carbonmonoxyhemoglobin with the shifts observed in paramagnetic cyanomethemoglobin provides information about the location of the acetyl derivatives relative to the heme irons. This approach is limited, however, by the lack of well defined structural information for the lysine residues on the protein surface. Additional tentative assignments have also been made, using the above approaches.     

Epistasis Creates Invariant Sites and Modulates the Rate of Molecular Evolution

Invariant sites are a common feature of amino acid sequence evolution. The presence of invariant sites is frequently attributed to the need to preserve function through site-specific conservation of amino acid residues. Amino acid substitution models without a provision for invariant sites often fit the data significantly worse than those that allow for an excess of invariant sites beyond those predicted by models that only incorporate rate variation among sites (e.g., a Gamma distribution). An alternative is epistasis between sites to preserve residue interactions that can create invariant sites. Through computer-simulated sequence evolution, we evaluated the relative effects of site-specific preferences and site-site couplings in the generation of invariant sites and the modulation of the rate of molecular evolution. In an analysis of ten major families of protein domains with diverse sequence and functional properties, we find that the negative selection imposed by epistasis creates many more invariant sites than site-specific residue preferences alone. Further, epistasis plays an increasingly larger role in creating invariant sites over longer evolutionary periods. Epistasis also dictates rates of domain evolution over time by exerting significant additional purifying selection to preserve site couplings. These patterns illuminate the mechanistic role of epistasis in the processes underlying observed site invariance and evolutionary rates.     

Protein fold and structure in the truncated (2/2) globin family

Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered "truncated hemoglobin" family. The analysis here presented, in agreement with previous surveys, shows that truncated hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 alpha-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 alpha-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the "truncated hemoglobins" term, that does not represent properly the observed 2-on-2 alpha-helical sandwich fold, and adopting the simple "2/2Hb" term to concisely address this protein family.     

Hemoglobin Athens-Georgia, or alpha 2 beta 2 40(C6)Arg replaced by Lys, a hemoglobin variant with an increased oxygen affinity.

A new hemoglobin variant, termed hemoglobin Athens-Georgia, has been found in a 23-year-old Caucasian student and three members of her family. The electrophoretic mobility of this variant at pH 9.0 is slightly less than that of hemoglobin-A. Arginyl residue in position 40 of the beta chain, corresponding to position 6 of the C helix, has been replaced by a lysyl residue. This amino acid substitution is at the alpha1-beta2 contact and slightly affects the oxygen binding properties of the hemoglobin molecule. Hemoglobin Athens-Georgia has an increased affinity for oxygen, a normal heme-heme interaction and a normal Bohr effect. Hematological abnormalities are not associated with this variant.     

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.     

Multiplicity of interactions between dromedary hemoglobin and solvent components. A structural and functional study

The interaction of dromedary hemoglobin with various solvent components [2-(p-chlorophenoxy)-2-methylpropionic acid (CFA), 2,3-bisphospho-D-glycerate (glycerate-2,3-P2) and chloride] has been studied. 1. CFA greatly lowers the oxygen affinity of dromedary hemoglobin. 2. The oxygen-linked CFA binding sites are probably located in the deoxy derivative at the alpha cleft, while in the oxy form and in the presence of two other effectors (glycerate-2,3-P2 and chloride) additional, structurally and possibly functionally relevant binding site(s) should be considered. 3. Both CFA and glycerate-2,3-P2 stabilize the deoxy-like tertiary structure in the oxy derivative. 4. Chloride appears to be fundamental to obtain quaternary structural changes. 5. Interaction energy, retained in the protein when the three ligands (CFA, glycerate-2,3-P2 and chloride) are bound to the oxy form, favours intermediates not stable if only one or two allosteric effector(s) is (are) present on the protein. 6. The oxygen affinity appears to be related to both tertiary and quaternary structural changes, while cooperatively is largely invariant with solvent conditions. In conclusion, the functional properties of dromedary hemoglobin do not depend in any simple way on the variety of stabilized conformations.     

Effect of anions on the oxygen binding properties of the hemoglobin components from trout (Salmo irideus).

The effect of several anions on the oxygen equilibrium of hemoglobin components (Hb Trout I, II, and IV) from trout has been investigated.The functional properties of Hb Trout I and II are very slightly affected by organic phosphates (ATP, IHP) and pyridoxal phosphate. On the other hand the oxygen affinity of both components is affected, to the same extent, by the presence of sodium chloride; this effect seems to be pH and temperature independent. For Hb Trout I experiments on the effect of orthophosphate, pyrophosphate and pyridoxal phosphate point to a certain degree of correlation between the size of the phosphate and its effect on the functional behavior of the protein.In the case of Hb Trout I and II the differences in the effect of the various organic and inorganic phosphates may be interpreted, at a molecular level, in terms of loss of charge complementarity and (or) steric hindrance effects.On the other hand, as in the case of human hemoglobin, organic or inorganic phosphates decrease the oxygen affinity of Hb Trout IV. In addition various phosphates shift the region where the Root effect is operative toward higher pH values, thereby acting as allosteric effectors. For pyridoxal phosphate, kinetic experiments have shown that the rate of binding to Hb trout IV is several orders of magnitude smaller than that for other organic phosphates, similarly to what has been reported for human hemoglobin.     

The role of a conserved histidine residue, His324, in Trigonopsis variabilisd-amino acid oxidase

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.     

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with 13C and determination and interpretation of terminal pK values

The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.     

Modulation of cellular protein trafficking by human immunodeficiency virus type 1 Nef: role of the acidic residue in the ExxxLL motif

The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E(160)xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins.     

Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the beta-carboxyvinyl and alpha-hydroxy-beta-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.     

Pre- and perinatal oxygen transport in mammals: the embryonic hemoglobins of the domestic pig (Sus scrofa domestica)

Pigs express two globin genes of beta-type, the epsilon- and chains during their embryonic stage. With the alpha- and epsilon-chains they form four embryonic hemoglobins. We describe here in detail the experimental procedures for sequencing the epsilon- and chains from the pig (Sus scrofa domestica) and we discuss the data with respect to their special functional properties. From the components Gower I and Heide I, we obtained all embryonic chains by chromatography on CM-Cellulose. The sequence of the tryptic peptides of the beta-type chains was established by automatic Edman-Begg degradation. They were aligned by comparison with the corresponding human chains. The epsilon-chains from man and pig differ in 20 positions, porcine and epsilon-chains only in 4 positions. Up to now these have only been found in pigs. A fetal hemoglobin (gamma-chains) was not observed. As a result of this work the sequences of all peptide chains of pig's hemoglobin are determined.     

Arenicola marina extracellular hemoglobin: a new promising blood substitute

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.