Altered Localization of the δ Subunit of the GABAA Receptor in the Thalamus of α4 Subunit Knockout Mice

The α4 subunit of the GABA_A receptor (GABA_AR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABA_AR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABA_AR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.     

Impact of Subunit Composition on the Uptake of α-Crystallin by Lens and Retina

Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in lens and retina. Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on α-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.     

Localization of α-galactomannan on the surface of Schizosaccharomyces pombe cells by scanning electron microscopy

Galactomannan was localized by scanning and transmission electron microscopy on the cells and cell walls of Schizosaccharomyces pombe. The markers were prepared from colloidal gold granules labelled with an -galactopyranosyl-binding lectin isolated from the seeds of Bandeiraea simplicifolia. Part or all of this -galactomannan was present in the outer layer of the cell wall and was uniformly distributed even on the fission scars.Non-Standard Abbreviations Au lectin-labelled colloid - SEM scanning electron microscopy - TEM transmission electron microscopy     

Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer_Bf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer_Bf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.     

Differences between α- and β-Chain Mutants of Human Haemoglobin and between α- and β-Thalassaemia. Possible Duplication of the α-Chain Gene

Human adult haemoglobin consists of two unlike pairs of polypeptide chains, and can be described as α₂β₂. Amino-acid substitutions in either of the two types of chain result in α- and β-chain variants. In thalassaemia, which causes a lowered production of haemoglobin, the α or the β chain can be affected, the result being α- or β-thalassaemia. There is a quantitative difference in the proportion of α- and β-chain variants to normal haemoglobin in the respective heterozygotes, and there is also a difference in the pattern of inheritance of α- and β-thalassaemia: these could possibly be explained by assuming that man has one gene for the β- and two for the α-chain.     

Localization of a Wide-Ranging Panel of Antigens in the Rat Retina by Immunohistochemistry: Comparison of Davidson��s Solution and Formalin as Fixatives

The preferred fixative for whole eyes is Davidson’s solution, which provides optimal tissue preservation while avoiding retinal detachment. Hitherto, the compatibility of Davidson’s solution with immunohistochemistry has been largely untested. The goal of the present study was to compare the immunolabeling patterns of a wide-ranging panel of commercially available, previously validated antibodies in formalin- and Davidson’s-fixed retinas. Immunohistochemistry was performed in normal pigmented rat eyes and, to facilitate localization of inducible proteins, eyes injected with the bacterial toxin lipopolysaccharide or subjected to laser-induced photoreceptor damage. Specificity of labeling was judged by the morphology and distribution of immunopositive cells, by the absence of signal in appropriate controls, and by comparison with expected staining patterns. Retinas fixed in formalin displayed only adequate morphological integrity but were highly compatible with all 39 antibodies evaluated. Retinas fixed in Davidson’s solution displayed morphological integrity superior to those fixed in formalin. Generally, the cellular and subcellular patterns and intensities of immunoreactivities obtained with each fixative were identical; however, Davidson’s fixative was less compatible with certain antibodies, such as the neurotransmitter γ-aminobutyric acid, the microglial marker iba1, the macroglial stress protein nestin, and the small heat shock proteins Hsp27 and αB-crystallin, shortfalls that somewhat temper enthusiasm concerning its use.     

Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Localization of α-tocopherol in hepatic chromatin from rats maintained on a tocopherol-deficient diet

• 1.1. A very significant amount of α-tocopherol was localized in hepatic chromatin when [³H]d-α-tocopherol was intravenously injected into rats maintained on tocopherol-deficient diet.
• 2.2. The site of association of α-tocopherol in the intrachromatin domain was examined by several methods.
• 3.3. The results show that α-tocopherol is predominantly localized in the heterochromatin and is associated with proteins exhibiting the characteristics of nonhistone proteins that have very high affinity for DNA.

     

Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase

DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.     

Variants of the α6 β1 Laminin Receptor in Early Murine Development: Distribution,Molecular Cloning and Chromosomal Localization of the Mouse Integrin α6 Subunit

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the α₆ subunit of murine α₆β₁ and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The α₆ subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than α₆ and has a more restricted distribution until the late blastocyst stage. α₆β₁ is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin α₆ subunit from a mammary gland γt11 library showed, as in man, an open reading frame encoding two variants of α₆, α₆ and α_6B. The identity of the α₆ amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine α₆ was mapped to chromosome 2. While undifferentiated ES and EC cells express only α_6B, α_6A is co-expressed in ES cells after differentiation is induced by retinoic acid. α_6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both α_6A and α_6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Identification of an 80kD Protein Associated with the α3β1 Integrin as a Proteolytic Fragment of the α3 Subunit: Studies with Human Keratinocytes

We have characterised a protein of approximately 80kD previously observed to co-immunoprecipitate with the α₃β₁ integrin in lysates of surface labelled human epiderrnalkerati-nocytes. The 80kD protein only appeared when keratinocytes were harvested with trypsin/EDTA prior to lysis and a protein of similar molecular mass could be immunoprecipitated from human dermal fibroblasts following treatment of the cells with trypsin/EDTA. N terminal sequencing established that the 80kD protein had homology with the as integrin subunit. Peptide-mass fingerprinting was used to confirm that the protein comprised the amino terminus of α₃ and established that the site of cleavage was after amino acid 629. The 80kD fragment could be coimmunoprecipitated with α₃β₁ using an antibody to the cytoplasmic domain of the α₃ subunit, showing that the fragment remained complexed with intact α₃β₁. When antibodies to the cytoplasmic and extracellular domains of α₃ were used to label human epidermis by immunofluorescence, the staining patterns were indistinguishable and there is therefore no evidence that proteolysis of α₃ plays a role in keratinocyte detachment from the basement membrane during terminal differentiation. Whether the 80kD fragment has any effects, positive or negative, on α₃β₁-mediated adhesion remains to be determined.     

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer

Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.     

Localization by site-directed mutagenesis of a galantamine binding site on α7 nicotinic acetylcholine receptor extracellular domain

Galantamine is an approved drug treatment for Alzheimer’s disease. Initially identified as a weak cholinesterase inhibitor, we have established that galantamine mainly acts as an ‘allosterically potentiating ligand (APL)’ of nicotinic acetylcholine receptors (nAChR). Meanwhile other ‘positive allosteric modulators (PAM)’ of nAChR channel activity have been discovered, and for one of them a binding site within the transmembrane domain has been proposed. Here we show, by performing site-directed mutagenesis studies of ectopically expressed chimeric chicken α7/mouse 5-hydroxytryptamine 3 receptor-channel complex, in combination with whole-cell current measurements, in the presence and absence of galantamine, that the APL binding site is different from the proposed PAM binding site. We demonstrate that residues T197, I196, and F198 of ß-strand 10 represent major elements of the galantamine binding site. Residue K123, earlier suggested as being ‘close to’ the APL binding site, is not part of this site but rather appears to play a role in coupling of agonist binding to channel opening and closing. Our data confirm our earlier results that the galantamine binding site is different from the ACh binding site. Both sites are in close proximity and hence may influence each other in a synergistic fashion. Other interesting areas identified in the present study are a ‘hinge’ region around and containing residues F122, K123, and K143 possibly being involved in relaying the signal of agonist binding to gating of the transmembrane channel, and a ‘folding centre’, with P119 as the dominating residue, that crucially positions the agonist binding site with respect to the hinge region.     

Localization by site-directed mutagenesis of a galantamine binding site on α7 nicotinic acetylcholine receptor extracellular domain

Galantamine is an approved drug treatment for Alzheimer's disease. Initially identified as a weak cholinesterase inhibitor, we have established that galantamine mainly acts as an 'allosterically potentiating ligand (APL)' of nicotinic acetylcholine receptors (nAChR). Meanwhile other 'positive allosteric modulators (PAM)' of nAChR channel activity have been discovered, and for one of them a binding site within the transmembrane domain has been proposed. Here we show, by performing site-directed mutagenesis studies of ectopically expressed chimeric chicken α7/mouse 5-hydroxytryptamine 3 receptor-channel complex, in combination with whole-cell current measurements, in the presence and absence of galantamine, that the APL binding site is different from the proposed PAM binding site. We demonstrate that residues T197, I196, and F198 of ?-strand 10 represent major elements of the galantamine binding site. Residue K123, earlier suggested as being 'close to' the APL binding site, is not part of this site but rather appears to play a role in coupling of agonist binding to channel opening and closing. Our data confirm our earlier results that the galantamine binding site is different from the ACh binding site. Both sites are in close proximity and hence may influence each other in a synergistic fashion. Other interesting areas identified in the present study are a 'hinge' region around and containing residues F122, K123, and K143 possibly being involved in relaying the signal of agonist binding to gating of the transmembrane channel, and a 'folding centre', with P119 as the dominating residue, that crucially positions the agonist binding site with respect to the hinge region.     

Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSP^WT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.     

α-Neurotoxin binding to acetylcholine receptor: Localization of the full profile of the cobratoxin-binding regions on the α-chain ofTorpedo californica acetylcholine receptor by a comprehensive synthetic strategy

Eighteen consecutive uniform overlapping synthetic peptides that spanned the entire extracellular part (residues 1–210) of the α-chain ofTorpedo californica acetylcholine receptor were screened for binding activity of¹²⁵I-labeled cobratoxin. Five toxin-binding regions were localized within residues 1–10, 32–41, 100–115, 122–150, and 182–198. The five toxin-binding regions may be distinct sites or, alternatively, different faces in one or more sites.     

Localization of a Single Transglutaminase-Reactive Glutamine in the Third Domain of RAP, the α2-Macroglobulin Receptor-Associated Protein

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln²⁶¹ acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.     

HbS-Savaria: The Anti-polymerization Effect of a Single Mutation in Human α-chains

Recombinant α-Savaria globin (α^S49R) was assembled with β^S chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (α₂^S49Rβ₂^E6V) that exhibited normal O₂ affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O₂ affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 ± 0.025 vs. 0.46 ± 0.03, respectively). The C_SAT of HbS increased from 16.7 ± 0.8 to 27.0 ± 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at α-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory α-chains and gene therapy of sickle cell anemia.