Alpha Beta Interface Research Articles

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface.

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain.

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the alpha1beta1 and the alpha1beta2 interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the alpha1beta1 and alpha1beta2 interfaces as reflected by 1H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment alpha(28-35) is in a helical conformation, while the segment alpha(19-22) is the nonhelical AB region. The shortened B-helix conserves the interactions of alpha1beta1 interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of alpha(23-26) without perturbing its overall global conformation.

Structure of the mitochondrial ATP synthase by electron cryomicroscopy.

We have determined the structure of intact ATP synthase from bovine heart mitochondria by electron cryomicroscopy of single particles. Docking of an atomic model of the F1-c10 subcomplex into a major segment of the map has allowed the 32 A resolution density to be interpreted as the F1-ATPase, a central and a peripheral stalk and an FO membrane region that is composed of two domains. One domain of FO corresponds to the ring of c-subunits, and the other probably contains the a-subunit, the transmembrane portion of the b-subunit and the remaining integral membrane proteins of FO. The peripheral stalk wraps around the molecule and connects the apex of F1 to the second domain of FO. The interaction of the peripheral stalk with F1-c10 implies that it binds to a non-catalytic alpha-beta interface in F1 and its inclination where it is not attached to F1 suggests that it has a flexible region that can serve as a stator during both ATP synthesis and ATP hydrolysis.

Constitutive analysis of the high-temperature deformation of Ti–6Al–4V with a transformed microstructure

The high-temperature deformation mechanisms of Ti–6Al–4V with a transformed microstructure were determined within the framework of inelastic-deformation theory. For this purpose, load-relaxation tests were conducted on samples with a lamellar structure containing different alpha-platelet thicknesses at temperatures of 715–900 °C. The flow stress-versus-strain rate curves for all the microstructures were well fit with an inelastic-deformation equation describing grain-matrix deformation (GMD) (dislocation glide+dislocation climb). However, for heavily pre-deformed specimens, grain-boundary sliding (GBS) as well as GMD was evident. The GBS rate was found to be most rapid for the microstructure with the thinnest alpha laths/platelets. Softening of heavily deformed material was attributed to a decrease in the internal-strength variable σ ∗ associated with reduced alpha–beta interface strength and to the occurrence of GBS.

Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state.

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.

Probing the conformation of hemoglobin presbyterian in the R-state.

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the alpha1beta2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(beta). In short, Lys-108(beta) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.

Conformational Changes in Hemoglobin S (βE6V) Imposed by Mutation of the βGlu7–βLys132 Salt Bridge and Detected by UV Resonance Raman Spectroscopy

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin.

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.

Crystal Structures of Deoxy- and Carbonmonoxyhemoglobin F1 from the Hagfish Eptatretus burgeri

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces.

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.

Avian haemoglobins and structural basis of high affinity for oxygen: structure of bar-headed goose aquomet haemoglobin.

Haemoglobin from the bar-headed goose (Anser indicus) has higher oxygen affinity than that from its lowland relatives such as greylag goose (A. anser). The crystal structure of bar-headed goose aquomet haemoglobin was determined at 2.3 A resolution and compared with the structures of the goose oxy, human, horse and other avian haemoglobins and the sequences of other avian haemoglobins. Four amino-acid residues differ between greylag goose and bar-headed goose haemoglobins, among which Alaalpha119 and Aspbeta125 in bar-headed goose haemoglobin reduces the contacts between the alpha(1) and beta(1) subunits compared with Pro and Glu, respectively, and therefore may increase the oxygen affinity by loosening the alpha(1)beta(1) interface. Compared with human oxy haemoglobin, the relative orientation of two alphabeta dimers in the bar-headed goose aquomet and oxy Hbs are rotated by about 4 degrees, indicating a unique quaternary structural difference from the typical R state. This new 'R(H)' state is probably correlated with the higher oxygen affinity of bar-headed goose haemoglobin.

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.

Lamprey Hemoglobin: STRUCTURAL BASIS OF THE BOHR EFFECT

Lampreys, among the most primitive living vertebrates, have hemoglobins (Hbs) with self-association and ligand-binding properties very different from those that characterize the alpha(2)beta(2) tetrameric Hbs of higher vertebrates. Monomeric, ligated lamprey Hb self-associates to dimers and tetramers upon deoxygenation. Dissociation to monomers upon oxygenation accounts for the cooperative binding of O(2) and its pH dependence. Honzatko and Hendrickson (Honzatko, R. B., and Hendrickson, W. A. (1986) Proc. Natl. Acad. Sci. U. S. A 83, 8487-8491) proposed that the dimeric interface of the Hb resembles either the alpha(1)beta(2) interface of mammalian Hbs or the contacts in clam Hb where the E and F helices form the interface. Perutz (Perutz, M. F. (1989) Quart. Rev. Biophys. 2, 139- 236) proposed a version of the clam model in which the distal histidine swings out of the heme pocket upon deoxygenation to form a bond with a carboxyl group of a second monomer. The sedimentation behavior and oxygen equilibria of nine mutants of the major Hb component, PMII, from Petromyzon marinus have been measured to test these models. The results strongly support a critical role of the E helix and the AB corner in forming the subunit interface in the dimer and rule out the alpha(1)beta(2) model. The pH dependence of both the sedimentation equilibrium and the oxygen binding of the mutant E75Q indicate that Glu(75) is one of two groups responsible for the Bohr effect. Changing the distal histidine 73 to glutamine almost completely abolishes the self-association of the deoxy-Hb and causes a large increase in O(2) affinity. The recent x-ray crystallographic determination of the structure of deoxy lamprey Hb, reported after the completion of this work (Heaslet, H. A., and Royer, W. E. (1999) Structure 7, 517-526), shows that the dimer interface does involve the E helix and the AB corner, supporting the measurements and interpretations reported here.

Molecular modelling ofTrematomus newnesi Hb 1: Insights for a lowered oxygen affinity and lack of root effect

Three-dimensional structural models of the hemoglobin (Hb 1) of the Antarctic fish Trematomus newnesi were built by homology modelling, using as template the X-ray crystallographic structures of Trematomus (previously named Pagothenia) bernacchii Hb 1, both in R and T state. The Hbs of these two fishes, although showing remarkably different oxygen binding properties, differ only by 4 residues in the alpha chain (142 aa) and 10 residues in the beta chain (146 aa). T. newnesi Hb1 R-state model, essentially performed as a quality control of the adopted modelling procedure, showed a good correspondence with the crystallographic one. Modelling of T. newnesi Hb1 in the T state was performed taking into account that the proton uptake by aspartate residues, proposed to be responsible for half of the Root effect in T. bernacchii Hb 1 (showing sharp pH dependent oxygen affinity and T-state overstabilization at low pH, i. e. Bohr and Root effect), does not occur in T. newnesi Hb1 (having nearly pH-independent lower oxygen affinity). Comparison with the template structure (submitted to the same minimization procedure) indicates that, in T. newnesi Hb1 T-state model, the substitution of Ile for Thr in 41 C6, in central position of the switch region, induces at the alpha(1)beta(2) interface structural modifications able to hamper the protonation. Similar modifications are also found in T. bernacchii Hb 1 modelled in the T state with the single substitution Thr-->Ile in 41alpha. These models also suggest that the lower oxygen affinity observed in T. newnesi Hb1 is related to structural differences at the alpha(1)beta(2) interface leading to a more stable low-affinity T state. Proteins 2000;39:155-165.

Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related.

To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity.

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin α and β chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation.

The interaction of heme-free alpha (alpha(o)) and heme-containing beta (beta(h)) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at 5 degrees C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10(7) M(-1) s(-1). This was 100-fold more rapid than the reported rate of combination of alpha(h) and beta(h) proteins. This encounter-like rate of semi-beta-hemoglobin (alpha(o)beta(h)) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB-GH helical entities. Molecular graphic analysis of modeled alpha(o) protein superimposed over native alpha(h) protein revealed an apo Mb-like structure with well-defined AB-GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the alpha1beta1 interface, was apparently responsible for the enhanced association properties of the alpha(o) protein.

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A.

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.

Dominant β‐thalassaemia: a highly unstable haemoglobin is caused by a novel 6 bp deletion of the β‐globin gene

Beta-thalassaemia is inherited as an autosomal recessive trait in most families. Particular interest has recently been focused on the molecular pathology of the rare forms with a dominant mode of inheritance. The index patient and her mother, who are described in this report, displayed typical clinical and haematological features of beta-thalassaemia intermedia with significant ineffective erythropoiesis and additional peripheral haemolysis. Molecular analysis demonstrated a heterozygous genotype for a novel 6 bp (TGGTCT) deletion of the beta-globin gene involving codons 33-35. This deletion results in the removal of two valine residues from the beta-globin chain at position 33/34 (B15/B16) and the substitution of the tyrosine residue at position 35 (C1) by an aspartic acid (beta 33-35 [B15-C1] Val-Val-Tyr-->0-0-Asp). According to the index patient's place of birth, this abnormal haemoglobin has been termed Hb Dresden. The stability of the variant and the normal beta-globin chains were similar during the incubation period of in vitro globin chain synthesis analysis. However, Hb Dresden is exquisitely unstable and cannot be detected in the peripheral blood by haemoglobin electrophoresis, high-performance liquid chromatography (HPLC) or isoelectric focusing. This instability can be explained by the vital structural role of the three affected amino acids that, in normal haemoglobin, establish a total of nine intermolecular bonds (five hydrophobic and four polar) at both the alpha1beta1 (alpha2beta2) and the alpha1beta2 (alpha2beta1) interface.