Contact inhibition within hemoglobin S polymer by thiol reagents

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.     

Heterogeneous nucleation in sickle hemoglobin: experimental validation of a structural mechanism

Sickle hemoglobin polymerizes by two types of nucleation: homogeneous nucleation of aggregates in solution, and heterogeneous nucleation on preexisting polymers. It has been proposed that the same contact that is made in the interior of the polymer between the mutant site beta6 and its receptor pocket on an adjacent molecule is the primary contact site for the heterogeneous nucleus. We have constructed cross-linked hybrid molecules in which one beta-subunit is from HbA with Glu at beta6, and the other is from HbS with a Val at beta6. We measured solubility (using sedimentation) and polymerization kinetics (using laser photolysis) on cross-linked hybrids, and cross-linked HbS as controls. We find approximately 4000 times less heterogeneous nucleation in the cross-linked AS molecules than in cross-linked HbS, in strong confirmation of the proposal. In addition, changes in stability of the nucleus support a further proposal that more than one beta6 contact is involved in the homogeneous nucleus.     

Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA.     

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.     

A model for the sickle hemoglobin fiber using both mutation sites

The standard molecular model of the fiber of the sickle hemoglobin (HbS: beta6 Glu-->Val) has been revised to allow both beta6 mutation sites to participate in intermolecular contacts, rather than only one beta6 site as previously thought, for four molecules per 14-molecule fiber cross section. This structure accurately predicts the copolymerization of hybridized mixtures of HbS with HbA or HbC (beta6 Glu-->Lys), which could not be reconciled with prior models in which only half the beta6 sites were required for assembly. This model suggests new contacts within the fiber and raises the question of whether these cross-linked double strands could possess added stability important in such processes as nucleation.     

Linkage of interactions in sickle hemoglobin fiber assembly: inhibitory effect emanating from mutations in the AB region of the alpha-chain is annulled by a mutation at its EF corner

The AB and GH regions of the alpha-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala(19) and Ala(21) of the AB corner with more flexible Gly or rigid alpha-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of alpha-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (alphaAsn(78) --> His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of alphaGln(78), His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (alphaGln(78) --> His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His(78). Similar synergistic coupling, between alphaHis(78) and an intra-double-strand contact point (alpha16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with alpha78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.     

A role for the alpha 113 (GH1) amino acid residue in the polymerization of sickle hemoglobin. Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (alpha 16/23) located in the AB region of the alpha-chain

A cluster of amino acid residues located in the AB-GH region of the alpha-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, alphaLeu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (betaGlu-6-->Val, alphaLeu-113-->His) to ascertain the hitherto unknown role of the alpha113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C(sat)) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the alpha113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the alpha113 mutation was coupled individually with two established fiber contact sites (alpha16 and alpha23) located in the AB region of the alpha-chain: HbS(alphaLys-16-->Gln, alphaLeu-113-->His), HbS(alphaGlu-23-->Gln, alphaLeu-113-->His). The single mutants at alpha16/alpha23 sites were also engineered as controls. The C(sat) values of the HbS point mutants involving sites alpha16 or alpha23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C(sat) values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of alpha113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.     

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.     

Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant

Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.     

Role of the beta4Thr-beta73Asp hydrogen bond in HbS polymer and domain formation from multinucleate-containing clusters

Fiber formation and domain formation from deoxy-HbS as well as from beta4 and beta73 HbS variants were investigated after temperature jump using DIC microscopy to gain a basic understanding of the determinants involved. Oversaturated deoxy-HbS generated numerous 14-stranded fibers and formed ovoid-shaped, multispherulitic domains. Domain number increased linearly as a function of time. Oversaturated deoxy-alpha2beta2(E6V,T4S) also generated time-dependent, ovoid-shaped spherulitic domains like HbS and alpha 2beta2(E6V,D73H) in the deoxy form. In contrast, alpha 2beta2(E6V,T4Y) and HbC-Harlem (alpha2beta2(E6V,D73N)) in the deoxy form generated time-dependent, ball-shaped domains containing many straight, crystalline-like fibers without evidence of branching. Some of these domains formed large needlelike crystals after overnight incubation. The inhibitory effect on polymer formation by beta4Tyr in HbS was stronger than that by beta4Ser but weaker than that by beta73Asn or beta73Leu. In contrast, both deoxy- and oxy-alpha2beta2(E6V,T4V) promoted formation of tiny, disordered amorphous aggregates without a delay time like oxy-HbS, which is in contrast to formation after a delay time of needlelike fibers for alpha 2beta2(E6V,D73L). Solubilities for both deoxy- and oxy-alpha 2beta2(E6V,T4V) were similar to that of deoxy-alpha 2beta2(E6V,D73H) but approximately 10-fold lower than that of deoxy-HbS. These results suggest that the strength of the hydrogen bond between beta4Thr and beta73Asp and the balance between the hydrogen bond and beta6Val hydrophobic interactions in deoxy-HbS polymers control formation of different types of fibers in a single domain or lead to formation of disordered, non-nucleated amorphous aggregates. These results also lead to a model in which multinucleation rather than a single-nucleation event occurs in a single cluster to generate numerous fibers growing from a single domain.     

Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability

As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.     

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.     

Controlling ligand binding in myoglobin by mutagenesis.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.     

Chimeric hemoglobins--hybrids of human and swine hemoglobin: assembly and stability of interspecies hybrids.

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.     

Liquid-liquid phase separation in hemoglobins: distinct aggregation mechanisms of the beta6 mutants

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (beta6 Lys) compared to HbS (beta6 Val) and HbA (beta6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the beta6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.     

The heme-globin and dimerization equilibria of recombinant human hemoglobins carrying site-specific beta chains mutations

The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Crystal structures of HbA2 and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of HbA2 and identification of the ferrocyanide binding site in Hb

Hemoglobin A(2) (alpha(2)delta(2)) is an important hemoglobin variant which is a minor component (2-3%) in the circulating red blood cells, and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is beta-chain production failure, HbA(2) acts as the predominant oxygen deliverer. HbA(2) has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26K(beta)), formed as a result of the splice site mutation on exon 1 of the beta-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with beta-thalassemia, it causes E/beta-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA(2) and HbE at 2.20 and 1.74 A resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA(2) and HbE. Using the coordinates of R2 state HbA(2), we modeled the structure of T state HbA(2) which allowed us to address the structural basis of the antisickling property of HbA(2). Using the coordinates of the delta-chain of HbA(2) (R2 state), we also modeled the structure of hemoglobin homotetramer delta(4) that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among beta(4), gamma(4), and delta(4), we formed a hypothesis regarding the possible tetramerization pathway of delta(4). The crystal structure of a ferrocyanide-bound HbA(2) at 1.88 A resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.     

Dissecting the Energies that Stabilize Sickle Hemoglobin Polymers

Sickle hemoglobin forms long, multistranded polymers that account for the pathophysiology of the disease. The molecules in these polymers make significant contacts along the polymer axis (i.e., axial contacts) as well as making diagonally directed contacts (i.e., lateral contacts). The axial contacts do not engage the mutant β6 Val and its nonmutant receptor region on an adjacent molecule, in contrast to the lateral contacts which do involve the mutation site. We have studied the association process by elastic light scattering measurements as a function of temperature, concentration, and primary and quaternary structure, employing an instrument of our own construction. Even well below the solubility for polymer formation, we find a difference between the association behavior of deoxy sickle hemoglobin molecules (HbS), which can polymerize at higher concentration, in comparison to COHbS, COHbA, or deoxygenated Hemoglobin A (HbA), none of which have the capacity to form polymers. The nonpolymerizable species are all quite similar to one another, and show much less association than deoxy HbS. We conclude that axial contacts are significantly weaker than the lateral ones. All the associations are entropically favored, and enthalpically disfavored, typical of hydrophobic interactions. For nonpolymerizable Hemoglobin, ΔH_o was 35 ± 4 kcal/mol, and ΔS was 102.7 ± 0.5 cal/(mol−K). For deoxyHbS, ΔH_o was 19 ± 2 kcal/mol, and ΔS was 56.9 ± 0.5 cal/(mol−K). The results are quantitatively consistent with the thermodynamics of polymer assembly, suggesting that the dimer contacts and polymer contacts are very similar, and they explain a previously documented significant anisotropy between bending and torsional moduli. Unexpectedly, the results also imply that a substantial fraction of the hemoglobin has associated into dimeric species at physiological conditions.