William S. Brinigar

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-β-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic β-globin which can be folded in the presence of cyanohemin and native α-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.

Cysteines β93 and β112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces

Abstract Three variants of tetrameric human hemoglobin, with changes at the α 1 β 2 / α 2 β 1 -interface, at the α 1 β 1 / α 2 β 2 -interface, and at both interfaces, have been constructed. At α 1 β 2 / α 2 β 1 -interface the β 93 cysteine was replaced by alanine ( β C93A), and at the α 1 β 1 / α 2 β 2 -interface the β 112 cysteine was replaced by glycine ( β C112G). The α 1 β 2 interface variant, β C93A, and the α 1 β 1 / α 1 β 2 double mutant, β (C93A+C112G), were crystallized in the T-state, and the structures determined at 2.0 and 1.8A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the β 112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of β C112G are essentially those of human hemoglobin A. At the β 93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of β 146His, weakening the important interaction of this residue with β 94Asp. As a result, when Cl − is present in the solution, at a concentration 100mM, the Bohr effect of the two mutants carrying the β 93Cys→Ala substitution, β C93A and β (C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between β 94Asp and Cl − in the salt link with β 146His in T-state hemoglobin. These results point to an interplay between the β His146- β Asp94 salt bridge and the Cl − in solution regulated by the Cys present at position β 93, indicating yet another role of β 93 Cys in the regulation of hemoglobin function.

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the α1β2 interface by the mutation β(Cys112 → Gly) at the α1β1 interface

Abstract The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant β-chains, and mutant hemoglobins where the substitution was on the surface, β(Thr4 → Asp), in the heme pocket, β(Val67 → Thr), at the 2,3-DPG binding site, β(Val1 → Met + His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the α 1 β 2 1 interface, β(Cys93 → Ala), the association constant was decreased three-fold. Conversely, in a mutant at the α 1 β 1 interface, β(Cys112 → Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the α 1 β 1 interface indicates that structural changes at this interface can be propagated through the protein to the α 1 β 2 interface and, thereby, exert an effect on the allosteric equilibrium.

Properties of Human Hemoglobins with Increased Polarity in the α- or β-Heme Pocket CARBONMONOXY DERIVATIVES

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A 0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val → Thr mutation has a larger conformational effect on the β-subunits than on the α-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the α-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271–26278). These effects are attributed to a different electrostatic interaction between Oγ of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Oγ in the β-subunit heme pocket.

Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin DEOXY DERIVATIVES

The dynamic and functional properties of mutant deoxyhemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified α chain and β chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the α chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the β chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified α chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the α chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified β chains is not observed by x-ray crystallography.

Recombinant hemoglobins as artificial oxygen carriers.

This paper describes the approaches we have taken to construct a) mutant hemoglobins with different oxygen affinities, and b) mutant hemoglobins and myoglobins that polymerize to high molecular weight aggregates in an effort to prevent extravasation and the associated vasoactivity. In vivo testing indicates that exchange transfusion of polymeric hemoglobins in mice does not result in vasoactivity and that polymeric hemoglobins are effective oxygen carriers to ischemic tissues irrespective of their oxygen affinity and cooperativity.

Assembly of Human Hemoglobin STUDIES WITH ESCHERICHIA COLI-EXPRESSED α-GLOBIN

The α-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native β-chains. The functional and structural properties of the expressed α-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl− and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the α1β1 and α1β2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed α-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the α-globin refolds and incorporates heme in the absence of the partner β-chain. Although the CO binding kinetics of recombinant α-chains were the same as that of native α-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant α-chain assumes alternating conformations in the absence of β-chain and indicate that the isolated α-chain exhibits a higher degree of conformational flexibility than the α-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed α-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

Synthesis of a hemin with structurally equivalent propionic acid side chains: 2,4-dimethyldeuterohemin IX dimethyl ester☆

Recent investigations concerning the structure of metalloporphyrin complexes and the nature of their interaction with proteins, for example [ I] have emphasized the need for a simple symmetrical porphyrin, closely related to protoporphyrin, to. which either one or two Iigand bearing groups may be covalently attached. The familiar derivatives of protoporphyrin IX are unsuitable because the propionic acid groups, by which ligands are most conveniently attached by ester or amide linkage, are nonequivalent. Therefore synthetic derivatives having two different groups on the propionic acid side chains are composed of two structural isomen which cannot be easily separated_ The presence of the two isomers was thought to be responsibIe, at least in part, for difficulties we have encountered in obtaining crystalline protoheme derivatives suitable for X-ray crystallography. In order to eliminate this problem, we developed an improved synthesis for 2.4-dimethyIdeuteroporphyrin IX dimethyl ester (I). Fischer and Jordan 121 first synthesized this compound starting from substituted pyrroles, a tedious procedure for any laboratory not already possessing the requisite pyrrole derivatives. A more convenient route, starting with protoporphyrin dimethyl ester is shown in Eq. (1). Porphyrin I also has been synthesized in low yield by Chang [ 31 starting from deuterohemin. Protorporphyrin IX dimethyl ester (II) was first oxidized with osmium tetroxide to the bis-glycol (III) and subseqtiently with sodium periodate to 2,Pdiformyldeuteroporphyrin IX dimethyl ester (IV) as described by Sparatore and Mauzerall 141. Compound III was purified by column chromatography in chloroform methanol on silica gel (yield, 69%). Compound IV crystallized from the reaction mixture (yield, 80%) and was not further purified since only one component was evident on thin layer chromatography, its visible spectrum was identical to that reported 143 and it was found to slowly decompose on standing

[50] Functional properties of rat liver mitochondria immobilized on an alkylsilylated surface

Publisher Summary This chapter discusses functional properties of the rat liver mitochondria immobilized on an alkylsilylated surface. Cellular organelles have been traditionally studied as dilute aqueous suspensions to which substances can be conveniently added, but cannot be easily and quickly removed. Therefore, it has been difficult to simulate their dynamic cellular environment where the concentrations of many substances are continuously changing. For this reason, a technique was desired which would more closely correspond to the conditions in vivo and would permit a wider range of experimental flexibility. In a sense, organelles, such as mitochondria and chloroplasts, are stationary in vitro relative to a continuously changing medium, much the same as they would be in vitro affixed to a solid support within a flow system. The chapter explores methods for attaching membrane enclosed particles to a finely divided solid phase. The chapter shows that to date only the rat liver mitochondria have been studied to any appreciable extent by this method, although spinach chloroplasts and rat liver microsomes have also been immobilized on the same support employed for mitochondria.

Is the serine protease “charge relay system” a charge relay system?

Summary The catalytic mechanism of the serine proteases as currently conceived specifies that the charge relay system Asp is ionized at pHs where the enzymes are active. This choice is questioned on the basis that external anions are found associated with the charge relay system of both chymotrypsin and subtilisin at pHs below the charge relay system pK, and no evidence has been found for the presence of an associated cation at pHs above the charge relay system pK. An alternative catalytic mechanism is proposed wherein Nδl of the His imidazole is hydrogen bonded to a neutral Asp, and His-Nɛ2 functions to relay a proton from Ser-Oγ to the substrate in a manner analogous to proton transfer in ice.