Scaling Properties of Universal Tetramers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Binding and structure of tetramers in the scaling limit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification, crystallization and Patterson search of haemoglobin IV from the armoured catfish Liposarcus anisitsi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 Angstrom resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique.

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O-2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Angstrom resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

A complexa fisiopatologia dos episódios vasooclusivos na anemia falciforme

Hemolytic anemia and vasoocclusion are the cardinal clinical features of sickle cell anemia. Vasoocclusion is a complex process involving not only the polymerization of deoxygenated sickle hemoglobin tetramers, but also interactions between sickle erythrocytes, vascular endothelium, platelets, leukocytes, and plasma proteins. The increased adherence of sickle erythrocytes to endothelium has been implicated as an early step in vasoocclusion. Other researchers have focused on leukocytes and platelets which might also contribute to disturbed blood flow. Microvascular occlusion results in acute painful crises, whereas macrovascular occlusion seems to be the cause of organ failure. The anemia results from the markedly shortened circulatory survival of sickle erythrocytes, together with a limited erythropoietic response. The erythropoiesis increases intensively, but it is not enough to balance the increased rate of erythrocytes destruction to maintain normal levels of total erythrocytes and hemoglobin concentrations; mainly by the low oxygen affinity of hemoglobin S and increased 2,3-Diphosphoglycerate. It is very difficult to separate processes leading to anemia or to vasoocclusion. Understanding the involvement of multiple blood componentes in vasoocclusion may elucidate the clinical manifestations and complications of sickle cell anemia, and may give new insights into the preventive and curative therapy.

Tjon Lines and Scaling Limit in Four-Body Systems

The fixed-slope correlation between tetramer and trimer binding energies, observed by Tjon in the context of nuclear physics, is mainly a manifestation of the dominance of the two-nucleon force in the nuclear potential, which makes the four-body scale on the order of the three-body one. In a more general four-boson case, the correlation between tetramer and trimer binding energies has a non-fixed slope, which expresses the dependence on the new scale. The associated scaling function generates a family of Tjon lines. This conclusion relies on a recent study with weakly-bound four identical bosons, within a renormalized zero-range Faddeev-Yakubovsky formalism. © 2012 Springer-Verlag.

Universality in Four-Boson Systems

We report recent advances on the study of universal weakly bound four-boson states from the solutions of the Faddeev-Yakubovsky equations with zero-range two-body interactions. In particular, we present the correlation between the energies of successive tetramers between two neighbor Efimov trimers and compare it to recent finite range potential model calculations. We provide further results on the large momentum structure of the tetramer wave function, where the four-body scale, introduced in the regularization procedure of the bound state equations in momentum space, is clearly manifested. The results we are presenting confirm a previous conjecture on a four-body scaling behavior, which is independent of the three-body one. We show that the correlation between the positions of two successive resonant four-boson recombination peaks are consistent with recent data, as well as with recent calculations close to the unitary limit. Systematic deviations suggest the relevance of range corrections. © 2012 Springer-Verlag.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations. © 2012 Elsevier B.V.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with Tc value of 333.4±0.2K for oxy-HbGp, while two peaks with Tc values of 332.2±0.1 and 338.4±0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher Tc values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323K, presenting oligomeric dissociation at 333 (12±3% of tetramer and 88±5% of whole HbGp) and 343K (70±5% of monomer and 30±2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar Tc values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure. © 2012 Elsevier B.V.

Four-Boson Systems Close to a Universal Regime

Universal properties of weakly-bound four-boson systems near the scaling limit are discussed by considering recent results obtained from the solution of Faddeev-Yakubovsky (FY) equations, which confirm a previous conjecture on a four-body scale dependence. In the present contribution, within a discussion on our numerical results obtained for the binding energies of two consecutive tetramer states, we are analyzing the relative relevance of the two possible configurations of the four-body system. © 2013 Springer-Verlag Wien.

Estudos conformocionais de diversas proteínas em solução utilizando a técnica de espalhamento de raios-X à baixo ângulo-saxs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudos estruturais de uma lectina presente em sementes de Lotus tetragonolobus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of Rhinodrilus alatus hemoglobin (HbRa) and its subunits: Evidence for strong interaction with cationic surfactants DTAB and CTAC

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)