Effects of Substitutions of Lysine and Aspartic Acid for Asparagine at β108 and of Tryptophan for Valine at α96 on the Structural and Functional Properties of Human Normal Adult Hemoglobin:  Roles of α1β1 and α1β2 Subunit Interfaces in the Cooperative Oxygenation Process

Tsai, Chih-Hsuan; Shen, Tong-Jian; Ho, Nancy T.; Ho, Chien

doi:10.1021/bi990286o

Cited by 44 publications

(92 citation statements)

References 47 publications

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“…This result suggests that structural changes at the ␣ 1 ␤ 1 ͞␣ 2 ␤ 2 interfaces can propagate to the ␣ 1 ␤ 2 ͞␣ 2 ␤ 1 interfaces and thus can affect the allosteric equilibrium. Support for this suggestion has been provided by Ho and coworkers, who found that Asn␤108(G10) 3 Lys and Asn␤108(G10) 3 Glu substitutions at the ␣ 1 ␤ 1 ͞␣ 2 ␤ 2 interfaces favor the T state relative to the R state, at low temperature and in the presence of allosteric effectors (24). Moreover, these two amino acid substitutions have been shown to have profound effects on the Bohr effect and on the linkage between oxygen and chloride ions (24).…”

Section: Allosteric Free Energy Changes At His␣103(g10) and His␣122(hmentioning

confidence: 80%

“…Support for this suggestion has been provided by Ho and coworkers, who found that Asn␤108(G10) 3 Lys and Asn␤108(G10) 3 Glu substitutions at the ␣ 1 ␤ 1 ͞␣ 2 ␤ 2 interfaces favor the T state relative to the R state, at low temperature and in the presence of allosteric effectors (24). Moreover, these two amino acid substitutions have been shown to have profound effects on the Bohr effect and on the linkage between oxygen and chloride ions (24). Therefore, it appears that the role of the ␣ 1 ␤ 1 ͞␣ 2 ␤ 2 interfaces in the Hb function is not negligible.…”

Section: Allosteric Free Energy Changes At His␣103(g10) and His␣122(hmentioning

confidence: 80%

“…Therefore, these resonances represent specific conformational markers for the tetrameric ␣ 2 ␤ 2 Hb structure. The resonance at 12.0 ppm has been assigned to the side chain N 2 H group of His␣103(G10), which is hydrogen-bonded to the side chain carbonyl group of Gln␤131(H9) [or to the peptide carbonyl of Asn␤108(G10)] at the ␣ 1 ␤ 1 and ␣ 2 ␤ 2 interfaces (22)(23)(24). The resonance at 12.8 ppm has been assigned to the side chain N 2 H group of His␣122(H5), which forms a water-mediated intersubunit hydrogen bond with the side chain of Tyr␤35(C1) at the ␣ 1 ␤ 1 and ␣ 2 ␤ 2 interfaces (25).…”

Section: Resultsmentioning

confidence: 99%

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A signature of the T → R transition in human hemoglobin

Mihailescu

Russu

2001

Proc. Natl. Acad. Sci. U.S.A.

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Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are His␣103(G10) and His␣122(H5) in each ␣ subunit of hemoglobin. They are located at the ␣1␤1 and ␣2␤2 interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the ␤ chains. We have measured the solvent exchange rates of side chain protons of His␣103(G10) and His␣122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by Ϸ1.5 kcal͞(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric ␣1␤1 and ␣2␤2 interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.T he Hb molecule has been a paradigm for understanding the molecular events underlying biological function. Cooperative oxygen-binding and allosteric effects have been rationalized by global thermodynamic models that postulate states of the molecule with specific energetic features (1-3). In the two-state model proposed by Monod, Wyman, and Changeux (2), the two energetic states are the tense (T) and the relaxed (R) states. The two states differ from each other in their affinity for ligand and in the interactions between subunits. Binding of oxygen to the deoxygenated (deoxy)-T state occurs with low affinity. However, the binding of the ligand destabilizes the T state and makes it more likely to switch to the high-affinity oxygenated (oxy)-R state. Thus, as ligation proceeds, the gradual enrichment in Hb molecules in the R state accounts for cooperativity in ligand binding. The success of this model in explaining the function of Hb has promoted its general application to a large variety of systems, including allosteric protein systems and nucleic acidbinding proteins (reviewed in refs. 4 and 5). However, the structural basis of this thermodynamic model is not yet fully understood.The first insights into the molecular structures representing the T and R thermodynamic states of Hb have been provided by crystallographic results on deoxy and ligated Hb obtained by Perutz (6, 7). The deoxy-T and ligated-R structures maintain the same ␣ 1 ␤ 1 and ␣ 2 ␤ 2 interfaces within ␣␤ dimers but differ greatly at the ␣ 1 ␤ 2 and ␣ 2 ␤ 1 interfaces between the two d...

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Section: Allosteric Free Energy Changes At His␣103(g10) and His␣122(hmentioning

confidence: 80%

Section: Allosteric Free Energy Changes At His␣103(g10) and His␣122(hmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

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A signature of the T → R transition in human hemoglobin

Mihailescu

Russu

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Mutations of Asnb108 in the a 1 b 1 interface can result in lower oxygen affinity and normal cooperativity compared to HbA, indicating a role for this interface in the allosteric transition. 29,30 Also, mutation of Glnb131, the acceptor of an a 1 b 1 interface H-bond, perturbs another interfacial H-bond some 7 Å distance, possibly via a network of ordered water molecules. Hydrogen exchange of the histidine residues involved in these H-bonds differs significantly between CO-and deoxyHb.…”

Section: Intra-and Interdimer Dynamicsmentioning

confidence: 99%

Hemoglobin Site-mutants Reveal Dynamical Role of Interhelical H-bonds in the Allosteric Pathway: Time-resolved UV Resonance Raman Evidence for Intra-dimer Coupling

Balakrishnan

Tsai

et al. 2004

Journal of Molecular Biology

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“…8 the Perutz salt bridge model has accounted for a wide range of both structural and functional Hb properties, as long as the symmetry of the Hb tetramer was preserved and as long as the system was studied as the average of its ligation intermediates. Still, elements of the two-state model, which permits global quaternary rearrangement, but no sequential inter-subunit coupling, have frequently been questioned from a wide range of perspectives (9)(10)(11)(12)(13)(14)(15)(16). Systematic deviations from the two-state model become most apparent when the Hb system is resolved into its separate, partially ligated microstates, some of which are asymmetrically ligated (17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Ackers

Dalessio

Lew

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of ␣␤ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer-dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both ␣-or both ␤-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer-dimer contacts is not communicated across the dimer-dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T͞R) component.M any tertiary and quaternary conformational events important for catalysis and cooperativity in biological macromolecules are energetically unfavorable, and must be driven by favorable free energy of ligand binding. In the case of human hemoglobin (Hb), initial binding of O 2 to the heme Fe drives intrinsically unfavorable conformation change within the globin. The associated energetic ''penalty'' results in lower binding constants for initial ligation. However, penalties for the additional binding steps are progressively reduced, generating Hb's characteristic sigmoidal binding curve. Contributing to this curve are the partially ligated Hb intermediates, composed of two different subunit types (␣ and ␤) with four binding sites (␣ 1 , ␤ 1 , ␣ 2 , ␤ 2 ). Up to four labile ligands (O 2 ) are present on each tetramer in multiple configurations of site occupancy within at least two distinct quaternary structures (T and R). The challenge in understanding the mechanism of Hb cooperativity has been to identify and measure the separate energetic penalties for each O 2 binding step, and then to correlate individual penalties with specific structural events.To approach a resolution of this complex problem, energetic components of the system's ligation intermediates were evaluated with no prior assumptions as to their quaternary structures. Analysis of the thermodynamic linkages between dimertetramer assembly and O 2 binding permitted the ligand binding constant of the noncooperative free dimer to be used as the thermodynamic reference s...

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Effects of Substitutions of Lysine and Aspartic Acid for Asparagine at β108 and of Tryptophan for Valine at α96 on the Structural and Functional Properties of Human Normal Adult Hemoglobin: Roles of α₁β₁ and α₁β₂ Subunit Interfaces in the Cooperative Oxygenation Process

Cited by 44 publications

References 47 publications

A signature of the T → R transition in human hemoglobin

A signature of the T → R transition in human hemoglobin

Hemoglobin Site-mutants Reveal Dynamical Role of Interhelical H-bonds in the Allosteric Pathway: Time-resolved UV Resonance Raman Evidence for Intra-dimer Coupling

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

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