Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A<sub>0</sub> fully stripped of any anions

Seixas, Flávio Augusto Vicente; Azevedo, Walter Filgueira de; Colombo, Marcio F.

doi:10.1107/s0907444999009750

Cited by 21 publications

(12 citation statements)

References 17 publications

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“…This shifts the R/T equilibrium in favor of the T-state and consequently lowers the overall affinity to oxygen (8,13). The well known Bohr effect and results reported for Cl Ϫ , also influencing the oxygen affinity of the T-state (14,15), show that, in a broader sense, H ϩ and Cl Ϫ can also be considered as being members of the family of allosteric effectors.…”

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confidence: 93%

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Schay

Smeller

Tsuneshige

et al. 2006

Journal of Biological Chemistry

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The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R-and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl ؊ , 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (␣FeO 2 ) 2 -(␤Zn) 2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70 -200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl ؊ . We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.Hemoglobin (1) is a tetrameric protein, which plays a vital role in the transport of oxygen. It consists of two dimers of ␣ and ␤ subunits that reversibly bind and release oxygen (1). The description of this cooperative phenomenon has been most frequently derived from the Monod-Wyman-Changeux (MWC) 2 two-state allosteric model (2) that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer. These distinct states are the fully unliganded T-state and the fully ligated R-state. Szabo and Karplus (3) modified the two-state model incorporating the stereochemical mechanism suggested by Perutz (4) for the T to R switch, and introduced ligation-induced tertiary changes within the T-state. In this extended model (MWC-SK), it was proposed that cooperativity still works through a ligation-induced shift in the equilibrium of states T and R, but the model attributed importance in the conformational switch to certain changes at the inter-and intrasubunit interfaces.

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confidence: 93%

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Schay

Smeller

Tsuneshige

et al. 2006

Journal of Biological Chemistry

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“…Thus, HbCO A exhibits a large number of R-type crystal structures, differing in structural detail, depending on a number of factors including the crystallization conditions. There are also several X-ray crystallographic structures for deoxyHb A available in the Protein Data Bank [PDB accession numbers: 1A3N (15), 4HHB (16), 1HGA (17), 1KD2 (18), 1RQ3 (19), 1XXT (6), 1BZ0 (20), 1YHR (6), and 2DN2 (21)], which were crystallized under different temperature, pH, buffer, and salt conditions. A basic assumption in correlating protein structure and function is that the structure of a protein in the crystalline state is the same as that under physiological solution conditions.…”

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confidence: 99%

Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

et al. 2007

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We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15 N-1 H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). In order to have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1 H-15 N Jcouplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed and these changes are largely within the α 1 β 1 (or α 2 β 2 ) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.Human normal adult hemoglobin (Hb A), a heterotetrameric protein that transports oxygen from the lungs to tissues, has served as an excellent model for investigating the structurefunction relationship in multimeric, allosteric proteins. Hb A consists of four subunits, i.e., two identical α-chains of 141 amino acid residues each and two identical β-chains of 146 amino acid residues each; each subunit contains a heme group leading to a complex with a molecular weight of ~ 64.5 kDa. The heme iron atom undergoes a spin-state change in going from a highspin paramagnetic Fe +2 (S=2) in the deoxy or unligated form to a diamagnetic Fe +2 (S=0) in the ligated form (with O 2 or CO). The oxygenation of Hb A is regulated by interactions between the O 2 -binding sites (homotropic interactions) and interactions between individual amino acid residues in the protein molecule and various solutes (heterotropic interactions). Heterotropic effectors include hydrogen ions, chloride ions, carbon dioxide, inorganic phosphate ions, and organic phosphate ions [e.g., 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP)] and are known to modulate the oxygen affinity of hemoglobin. For reviews on the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript structure-function relationship on hemoglobin, see (1-3). Allosteric effectors, 2,3-BPG and IHP, exert a significant effect on the oxygen-binding pr...

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“…In the absence of crystallographic evidence for the structure of this intermediate affinity state of deoxy-Hb, Colombo and Seixas had defined it, operationally, as a P state. Recent crystallographic studies have revealed, however, that although the increase in O 2 -affinity and the increase in protein hydration accompanies the release of anions from deoxy-Hb, the T quaternary arrangement of the tetramer is preserved (Seixas et al, 1999). The existence of two T states of different O 2 affinities, predicted by Dn w measurements, has been confirmed more recently, by oxygen-binding experiments with Hb encapsulated in silica gel, which prevents the O 2 -induced T to R quaternary transition of Hb (Bruno et al, 2001;Shibayama and Saigo, 2001).…”

Section: Fig 1 Portrays the Closed Cell Used To Weight The Hydration Ofmentioning

confidence: 99%

The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin

Salvay

Grigera

Colombo

2003

Biophysical Journal

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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.

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Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A₀ fully stripped of any anions

Cited by 21 publications

References 17 publications

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin

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Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A0 fully stripped of any anions

Cited by 21 publications

References 17 publications

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin

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Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A₀ fully stripped of any anions