Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

Mozzarelli, Andrea; Rivetti, Claudio; Rossi, Gian Luigi; Eaton, William A.; Henry, Eric R.

doi:10.1002/pro.5560060230

Cited by 60 publications

(58 citation statements)

References 38 publications

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“…Data fit to a hyperbolic binding function yields a p50 (the oxygen pressure at half saturation) of 133.1±3.1 torr and a Hill coefficient of 0.89±0.02, indicating a non-cooperative binding in the T state. Oxygen affinity overlaps with that obtained in T state Hb crystals [218,219], and with K1 (the affinity constant for binding of the first oxygen molecule to the Hb tetramer) obtained in solution in the same experimental conditions [148]. Open circles: the oxygen binding curve for HbA in solution in 100 mM HEPES, 1 mM EDTA, pH 7, 15°C is shown for comparison (p50 = 2.29±0.03 torr, Hill coefficient = 2.54±0.10) [54].…”

Section: Green Fluorescent Proteinsupporting

confidence: 63%

“…However, the most striking result about Hb encapsulated in silica gels is the perfect conservation of equilibrium oxygen binding properties observed in solution under comparable conditions [54,57,58,82,100,101] (Fig. 3) [218,219], which legitimates the exploitation of this system in kinetic experiments aiming to test theoretical models of Hb allosteric regulation (see Section 4).…”

Section: Heme Proteinsmentioning

confidence: 92%

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Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Ronda

Bruno

Campanini

et al. 2015

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Abstract:The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques.

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Section: Green Fluorescent Proteinsupporting

confidence: 63%

Section: Heme Proteinsmentioning

confidence: 92%

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Ronda

Bruno

Campanini

et al. 2015

COC

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“…From the ratio of the binding constants measured for light polarized along orthogonal axes, which have different projections of the a and b hemes, Mozzarelli and coworkers estimated q and therefore that d T 5 3 (48,49). In the subsequent discussion we ignore this small effect, and assume the binding to both quaternary structures is perfectly non-cooperative.…”

Section: The Cooperon Model Of Brunorimentioning

confidence: 99%

Evolution of allosteric models for hemoglobin

et al. 2007

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SummaryWe compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)-and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond -millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.IUBMB Life, 59: 586-599, 2007

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“…The change in oxygen affinity caused by heterotropic effectors without a change in quaternary structure is readily explained by the TTS model as resulting from an alteration in the equilibrium between low-affinity t and high-affinity r tertiary conformations. Because lattice constraints prevent the tertiary conformational changes that occur in solution (17)(18)(19)37), solution NMR spectroscopy (38) will most probably be required to identify the functionally relevant structural differences between r and t. Finally, the results of our study suggest that it will be interesting to apply the TTS model to other systems in which neither the MWC nor sequential models adequately describe allosteric behavior (39,40).…”

mentioning

confidence: 95%

“…In sharp contrast to hemoglobin free in solution, O 2 binding to gel-encapsulated hemoglobin, like O 2 binding to the hemoglobin crystal (17)(18)(19), is noncooperative (Fig. 2).…”

mentioning

confidence: 99%

New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels

Viappiani

Bettati²,

Bruno³

et al. 2004

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

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108

141

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To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of the T quaternary structure has kinetic properties almost identical to those of subunits of the R quaternary structure. Addition of allosteric inhibitors reduces both the fraction of R-like subunits and the oxygen affinity of the T quaternary structure. These kinetic and equilibrium results are readily explained by a recently proposed generalization of the Monod-Wyman-Changeux model in which a preequilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.T he two-state allosteric model of Monod, Wyman, and Changeux (1) represented a conceptual breakthrough in explaining the cooperative and regulated behavior of multisubunit proteins, with application to a wide range of biological systems (2-5). Monod, Wyman, and Changeux proposed that ligands control protein function by altering a preexisting equilibrium between high (R) and low (T) reactivity conformations that differ in intersubunit bonding (quaternary structure) and not by inducing conformational changes that are propagated to neighboring subunits as in a sequential model (6, 7). Enzyme activation, for example, results from preferential binding of ligands to the R quaternary structure, whereas inhibitors preferentially bind to T. However, a long-known serious deficiency in the application of the Monod-Wyman-Changeux (MWC) model to hemoglobin, the paradigm of allosteric proteins, is that inhibitors may also change oxygen (O 2 ) affinity without a change in quaternary structure (8-11). To understand this phenomenon, we have investigated the ligand binding kinetics and equilibria of hemoglobin encapsulated in silica gels in either the T or R quaternary structure (Fig. 1).Previous studies of hemoglobin encapsulated in silica gels showed greatly simplified equilibrium properties, compared with those in solution, because quaternary conformational changes are markedly slowed by the constraints of the surrounding cross-linked polymer (12-16). In sharp contrast to hemoglobin free in solution, O 2 binding to gel-encapsulated hemoglobin, like O 2 binding to the hemoglobin crystal (17-19), is noncooperative (Fig. 2). Encapsulation as the fully deoxygenated molecule traps hemoglobin in the low-affinity T quaternary structure, whereas encapsulation as the fully oxygenated molecule traps hemoglobin in the high-affinity R structure (12). Moreover, the affinity of the deoxy-encapsulated molecule is lowered by inhibitor ligands (called negative heterotropic allosteric effectors) such as protons, chloride ions, inositol hexaphosphate, and bezafibrate (Fig. 2) in the ...

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Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

Cited by 60 publications

References 38 publications

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Evolution of allosteric models for hemoglobin

New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels

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