Allosteric Equilibrium Research Articles

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease.

Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography.

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.

Remote oxidative modifications induced by oxygen free radicals modify T/R allosteric equilibrium of a hyperthermophilic lactate dehydrogenase

L-Lactate dehydrogenase (LDH) is a model protein allowing to shed light on the fundamental molecular mechanisms that drive the acquisition, evolution and regulation of enzyme properties. In this study, we test the hypothesis of a link between thermal stability of LDHs and their capacity against unfolding induced by reactive oxygen species (ROS) generated by γ-rays irradiation. By using circular dichroism spectroscopy, we analysed that high thermal stability of a thermophilic LDH favours strong resistance against ROS-induced unfolding, in contrast to its psychrophilic and mesophilic counterparts that are less resistant. We suggest that a protein’s phenotype linking strong thermal stability and resistance against ROS damages would have been a selective evolutionary advantage. We also find that the enzymatic activity of the thermophilic LDH that is strongly resistant against ROS-unfolding is very sensitive to inactivation by irradiation. To address this counter-intuitive observation, we combined mass spectrometry analyses and enzymatic activity measurements. We demonstrate that the dramatic change on LDH activity was linked to remote chemical modifications away from the active site, that change the equilibrium between low-affinity tense (T-inactive) and high-affinity relaxed (R-active) forms. We found the T-inactive thermophilic enzyme obtained after irradiation can recover its LDH activity by addition of the allosteric effector 1, 6 fructose bis phosphate.We analyse our data within the general framework of allosteric regulation, which requires that an enzyme in solution populates a large diversity of dynamically-interchanging conformations. Our work demonstrates that the radiation-induced inactivation of an enzyme is controlled by its dynamical properties.

Residues W215, E217 and E192 control the allosteric E*-E equilibrium of thrombin

A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215–217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190–193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215–217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.

Probing prothrombin structure by limited proteolysis

Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.

Profiling Proteasome Activity By Porphyrins

Inspired by some preliminary data and their versatile chemistry we have been investigating the ability of porphyrins to inhibit proteasome activity. Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms. They may bind to the 20S proteasome gates hindering the entrance of the substrate into the catalytic chamber or, by functional connection between the α- and β-rings, allow for allosteric modulation. By contrast, anionic porphyrins may activate the core particle by facilitating the access of the substrate to the proteasome interior, likely regulating the “open”-“closed” allosteric equilibrium. In conclusion, the positioning of the charged substituents represents a sort of key code to fine-tuning porphyrins interference with proteasome gating.

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S.

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30nM and activating at (IDE) ≥ 30nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.

Allosteric mechanisms underlying the adaptive increase in hemoglobin-oxygen affinity of the bar-headed goose.

The high blood-O2 affinity of the bar-headed goose (Anser indicus) is an integral component of the biochemical and physiological adaptations that allow this hypoxia-tolerant species to undertake migratory flights over the Himalayas. The high blood-O2 affinity of this species was originally attributed to a single amino acid substitution of the major hemoglobin (Hb) isoform, HbA, which was thought to destabilize the low-affinity T state, thereby shifting the T-R allosteric equilibrium towards the high-affinity R state. Surprisingly, this mechanistic hypothesis has never been addressed using native proteins purified from blood. Here, we report a detailed analysis of O2 equilibria and kinetics of native major HbA and minor HbD isoforms from bar-headed goose and greylag goose (Anser anser), a strictly lowland species, to identify and characterize the mechanistic basis for the adaptive change in Hb function. We find that HbA and HbD of bar-headed goose have consistently higher O2 affinities than those of the greylag goose. The corresponding Hb isoforms of the two species are equally responsive to physiological allosteric cofactors and have similar Bohr effects. Thermodynamic analyses of O2 equilibrium curves according to the two-state Monod-Wyman-Changeaux model revealed higher R-state O2 affinities in the bar-headed goose Hbs, associated with lower O2 dissociation rates, compared with the greylag goose. Conversely, the T state was not destabilized and the T-R allosteric equilibrium was unaltered in bar-headed goose Hbs. The physiological implication of these results is that increased R-state affinity allows for enhanced O2 saturation in the lungs during hypoxia, but without impairing O2 delivery to tissues.

Interspecific variation and plasticity in hemoglobin nitrite reductase activity and its correlation with oxygen affinity in vertebrates

Deoxygenated hemoglobin (Hb) is a nitrite reductase that reduces naturally occurring nitrite to nitric oxide (NO), supplying physiological relevant NO under hypoxic conditions. The nitrite reductase activity is modulated by the allosteric equilibrium between the R and T structures of Hb that also determines oxygen affinity. In the present study we investigated nitrite reductase activity and O2 affinity in Hbs from ten different vertebrate species under identical conditions to disclose interspecific variations and allow an extended test for a correlation between the rate constant for nitrite reduction and O2 affinity. We also tested plastic changes in Hb properties via addition of T-structure-stabilizing organic phosphates (ATP and GTP). The decay in deoxyHb during its reaction with nitrite was exponential-like in ectotherms (Atlantic hagfish, carp, crucian carp, brown trout, rainbow trout, cane toad, Indian python and red-eared slider turtle), while it was sigmoid in mammals (harbor porpoise and rabbit). Typically, hypoxia-tolerant species showed a faster reaction than intolerant species. Addition of ATP and GTP decreased O2 affinity and slowed the rate of nitrite reduction in a concentration-dependent manner. The initial second order rate constant of the deoxyHb-mediated nitrite reduction showed a strong curvilinear correlation with oxygen affinity among all ectothermic vertebrates, and the relationship also applied to plastic variations of Hb properties via organic phosphates. The relationship predicts high nitrite reductase activity in hypoxic tolerant species with high Hb-O2 affinity and reveals that the decrease in erythrocyte ATP and/or GTP during acclimation to hypoxia in ectotherms increases the erythrocyte NO generating capacity.

The Simple and Unique Allosteric Machinery of Thermus caldophilus Lactate Dehydrogenase : Structure-Function Relationship in Bacterial Allosteric LDHs.

Many bacterial L-lactate dehydrogenases (LDH) are allosteric enzymes, and usually activated by fructose 1,6-bisphosphate (FBP) and often also by substrate pyruvate. The active and inactive state structures demonstrate that Thermus caldophilus, Lactobacillus casei, and Bifidobacterium longum LDHs consistently undergo allosteric transition according to Monod-Wyman-Changeux model, where the active (R) and inactive (T) states of the enzymes coexist in an allosteric equilibrium (pre-existing equilibrium) independently of allosteric effectors. The three enzymes consistently take on open and closed conformations of the homotetramers for the T and R states, coupling the quaternary structural changes with the structural changes in binding sites for substrate and FBP though tertiary structural changes. Nevertheless, the three enzymes undergo markedly different structural changes from one another, indicating that there is a high variety in the allosteric machineries of bacterial LDHs. L. casei LDH undergoes the largest quaternary structural change in the three enzymes, and regulates its catalytic activity though a large linkage frame for allosteric motion. In contrast, T. caldophilus LDH exhibits the simplest allosteric motion in the three enzymes, involving a simple mobile structural core for the allosteric motion. TcLDH likely mediates its allosteric equilibrium mostly through electrostatic repulsion within the protein molecule, providing an insight for regulation machineries in bacterial allosteric LDHs.

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.

Allosteric regulation: Illuminating allosteric conformational change with an environmentally sensitive fluorescent probe

Allosteric regulation was a hot topic in the 1960s, but there was very limited structural data on allosteric equilibria, and no solid information on the rates of allosteric conformational changes. In this Biochemical Journal Classic paper from 1969 George Radda and his first D.Phil. student, George Dodd determined the rate of allosteric transition in the regulatory enzyme glutamate dehydrogenase by a method new in the 1960s, the fluorescence of an environmentally sensitive extrinsic probe.

Realizing the recognition features of model antipsychotic compounds by important protein: Photochemical and computational studies

Phenothiazine and its derivatives are the most effective antipsychotic drugs. They have been used in the treatment of serious mental and emotional symptoms including bipolar disorder, organic psychoses, psychotic depression and schizophrenia. However, these drugs cause serious side effects such as akathisia, hyperprolactinaemia and neuroleptic malignant syndrome. In this work we investigated the molecular recognition of two typical phenothiazine compounds, phenosafranin and safranin O by the most pivotal heme protein hemoglobin using steady state and time-resolved fluorescence, extrinsic 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescent probe, circular dichroism (CD) along with computational modeling. Results show phenothiazines complex with protein via formation of adducts at 298K with moderate strengths of 3.555×104M−1 and 2.567×104M−1 for the hemoglobin–phenosafranin and hemoglobin–safranin O, respectively. We also found phenothiazines were effectors at the protein allosteric site, which affects the allosteric equilibrium. Further, time-resolved fluorescence and hydrophobic ANS experiments showed the static mechanism is dominated for the shrinkage in the fluorescence intensity of β-37 Trp residue at the α1β2 interface. The stoichiometric proportion of the protein–drug adduct is 1:1, as derived from Job’s plot. Several crucial noncovalent bonds, including hydrogen bonds, π–π stacking and hydrophobic interactions played a major role in stabilizing the noncovalent conjugates. Based on three-dimensional fluorescence, we concluded that the conformation of hemoglobin is partially destabilized after recognition with phenothiazines. These alterations were confirmed by far-UV CD spectra that showed the α-helix of protein decreased from 78.3% in free hemoglobin to 62.8% and 64.8% in hemoglobin–phenosafranin and hemoglobin–safranin O, respectively. Computer-aided molecular docking was consistent, indicating that both phenothiazines are situated within the pocket composed of α1 and β2 subunits. Affinity of hemoglobin to phenosafranin is superior compared with safranin O. This difference may be explained by the methyl group substituent on A- and C-rings, and by the different molecular volume between phenosafranin and safranin O. Our data provides further explanation of the overall pharmacokinetics of phenothiazines and sheds light on the allosteric regulation of heme proteins.

The Core of Allosteric Motion in Thermus caldophilusl-Lactate Dehydrogenase

For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.

Capturing the Hemoglobin Allosteric Transition in a Single Crystal Form

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

Measuring how much work the chaperone GroEL can do

Noncovalently "stacked" tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent cross-links provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.

Molecular and functional characterization of hemocyanin of the giant African millipede,Archispirostreptus gigas

In contrast to other terrestrial arthropods, where gaseous O2 that fuels aerobic metabolism diffuses to the tissues in tracheal tubes, and most other metazoans, where O2 is transported to tissues by circulating respiratory proteins, the myriapods (millipedes and centipedes) strikingly have tracheal systems as well as circulating hemocyanin (Hc). In order to elucidate the evolutionary origin and biological significance of millipede Hc, we report the molecular structure (subunit composition and amino acid sequence) of multimeric (36-mer) Hc from the forest floor-dwelling giant African millipede Archispirostreptus gigas and its allosteric oxygen-binding properties under various physico-chemical conditions. Archispirostreptus gigas Hc consists of only a single subunit type with differential glycosylation. Phylogenic analysis revealed that millipede Hc is a sister group to centipede HcA, which supports an early divergence of distinct Hc subunits in myriapods and an ancient origin of multimeric Hcs. Archispirostreptus gigas Hc binds O2 with a high affinity and shows a strong Bohr effect. O2 binding is, moreover, modulated by Ca(2+) ions, which increase the O2 affinity of the Hc in the tense (T; deoxygenated) as well as the relaxed (R; oxygenated) states, and by (l)-lactate, which modulates Hc-O2 affinity by changing the allosteric equilibrium constant, L. Cooperativity in O2 binding at half O2 saturation (n50) is pH dependent and maximal at ~pH 7.4, and the number of interacting O2-binding sites (q) is markedly increased by binding Ca(2+). The data are discussed in the light of the mutually supplementary roles of Hc and the tracheal system for tissue O2 supply.

A Novel Case of Co-Inheritance of Rare Hemoglobin Variant; Hb Rancho Mirage [βCd 143 (CAC→GAC) His→Asp] with Hb D-Punjab

AbstractAbstract 4759 IntroductionCo-inheritance of Hb Rancho Mirage and Hb D-Punjab produces a clinically significant condition through increasing oxygen affinity that causes a compensatory erythrocytosis. We report a first case of co-inheritance of Rancho Mirage with Hb D-Punjab which differs in its clinical presentation from that described in the literature. Case presentationA 59-year-old Pakistani male was admitted to hospital because of chest tightness. The imaging studies showed no marked organomegaly or masses. Laboratory investigations revealed erythrocytosis with high hematocrit and borderline MCV and MCH. Arterial blood gas testing (ABG) showed low pO2 and JAK2 V617F mutation was not detected. Erythropoietin level was normal. Hemoglobin analysis through high performance liquid chromatography (HPLC) revealed two hemoglobin variants; Hb D-Punjab (32.5%) and the remaining Hb comprised of a fast moving variant. The variant was faster than Hb F expressing at a considerably high level. There was no detectable Hb A. (figure 1, HPLC shows both variants)At the molecular level, the β globin gene was amplified and sequenced using specific primers. DNA analysis demonstrated the presence of two mutations in Exon 3. The first one was a rare mutation in βCd 143 (CAC→GAC) causing a substitution of His→Asp that was identified as Hb-Rancho Mirage. It is speculated that the replacement of the ‘His‘ residue with that of ‘Asp' would unequivocally alter the oxygen binding characteristics of the heme pocket. The other mutation was the common variant defined as Hb D-Punjab βCd 121 (GAA→CAA). (figure 2, DNA sequencing shows both variants). The patient had undergone regular venesection to alleviate erythrocytosis. DiscussionThe co-inheritance of Hb Rancho Mirage with Hb D-Punjab has never been reported previously. Both mutations exist in the β globin gene giving rise to concomitant beta chain variants. Carriers of Hb Rancho Mirage are anticipated with mild anemia. However, coexistence of both abnormal hemoglobins showed functional defects manifested as polycythemia, likely due to the disruption of the 2,3 DPG binding site which is actively involved in the allosteric equilibrium during oxygen transport.The current case depicts the first occurrence of a double heterozygosity for two beta chain variants affecting exon 3 of the β globin gene with a relatively mild to moderate phenotype. Family studies are recommended to understand further the structure and function relationship of this rare hemoglobin variant in association with a more common Hb D- Punjab. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure, Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves.

3I0912 ヘモグロビンのアロステリック平衡のリアルな描像(ヘム蛋白質,口頭発表)

3I0912 ヘモグロビンのアロステリック平衡のリアルな描像(ヘム蛋白質,口頭発表)