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

Viappiani, Cristiano; Bettati, Stefano; Bruno, Stefano; Ronda, Luca; Abbruzzetti, Stefania; Mozzarelli, Andrea; Eaton, William A.

doi:10.1073/pnas.0405987101

Cited by 108 publications

(158 citation statements)

References 42 publications

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“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

2006

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Spectrally-resolved infrared stimulated vibrational echo spectroscopy is used to measure the fast dynamics of heme-bound CO in carbonmonoxy-myoglobin (MbCO) and hemoglobin (HbCO) embedded in silica sol-gel glasses. On the time scale of ~100 fs to several ps, the vibrational dephasing of the heme-bound CO is measurably slower for both MbCO and HbCO relative to aqueous protein solutions. The fast structural dynamics of MbCO, as sensed by the heme-bound CO, are influenced more by the sol-gel environment than those of HbCO. Longer time scale structural dynamics (tens of ps), as measured by the extent of spectral diffusion, are the same for both proteins encapsulated in sol-gel glasses compared to aqueous solutions. A comparison of the sol-gel experimental results to viscosity dependent vibrational echo data taken on various mixtures of water and fructose shows that the sol-gel encapsulated MbCO exhibits dynamics that are the equivalent to the protein in a solution that is nearly 20 times more viscous than bulk water. In contrast, the HbCO dephasing in the sol-gel reflects only a 2-fold increase in viscosity. Attempts to alter the encapsulating pore size by varying the molar ratio of silane precursor to water (R-value) used to prepare the sol-gel glasses were found to have no effect on the fast or steady-state spectroscopic results. The vibrational echo data are discussed in the context of solvent confinement and protein-pore wall interactions to provide insights into the influence of a confined environment on the fast structural dynamics experienced by a biomolecule.

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Section: Introductionmentioning

confidence: 99%

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

2006

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“…Interest in Hb continues to the present, with new experimental and theoretical methods aimed at characterizing intermediate ligation states of the molecule (3)(4)(5) and at gaining a deeper understanding of its allosteric transition (6)(7)(8)(9)(10)(11).…”

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A quantum-chemical picture of hemoglobin affinity

Alcantara

Spiro

et al. 2007

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

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Understanding the molecular mechanism of hemoglobin cooperativity remains an enduring challenge. Protein forces that control ligand affinity are not directly accessible by experiment. We demonstrate that computational quantum mechanics/molecular mechanics methods can provide reasonable values of ligand binding energies in Hb, and of their dependence on allostery. About 40% of the binding energy differences between the relaxed state and tense state quaternary structures result from strain induced in the heme and its ligands, especially in one of the pyrrole rings. The proximal histidine also contributes significantly, in particular, in the ␣-chains. The remaining energy difference resides in protein contacts, involving residues responsible for locking the quaternary changes. In the ␣-chains, the most important contacts involve the FG corner, at the ''hinge'' region of the ␣1␤2 quaternary interface. The energy differences are spread more evenly among the ␤-chain residues, suggesting greater flexibility for the ␤-than for the ␣-chains along the quaternary transition. Despite this chain differentiation, the chains contribute equally to the relaxed substitute state energy difference. Thus, nature has evolved a symmetric response to the quaternary structure change, which is a requirement for maximum cooperativity, via different mechanisms for the two kinds of chains.allostery ͉ cooperativity ͉ heme ͉ quantum mechanics/molecular mechanics H emoglobin has been a paradigm for our understanding of multisubunit proteins, beginning with the pioneering work of Adair (1) and then with the elucidation of the oxygenated and deoxygenated crystal structures of Hb by Perutz (2), some four decades ago. Interest in Hb continues to the present, with new experimental and theoretical methods aimed at characterizing intermediate ligation states of the molecule (3-5) and at gaining a deeper understanding of its allosteric transition (6-11).The Hb molecule is composed of four chains that contain a heme group capable of reversibly binding CO or O 2 . The molecule is arranged as a dimer of dimers, where each dimer is made up of an ␣-and a ␤-chain (denoted as ␣ 1 ␤ 1 or ␣ 2 ␤ 2 ) with a hydrophobic intradimer interface. Adair observed that Hb binds oxygen in a cooperative manner, successive ligation events occurring with higher affinity. For some time it was believed that such cooperativity could be explained by direct interaction of the closely packed heme groups. However the 1960s saw the appearance of the first allosteric models for Hb cooperativity. In particular, the Monod-Wyman-Changeux (12) model postulated the existence of two states of the molecule: the relaxed (R) state with high ligand affinity and the tense (T) state with low affinity. Ligand binding preferentially stabilized the R form and led to cooperativity. Early Hb crystallization attempts revealed that deoxy crystals would shatter when exposed to oxygen, which suggested that a quaternary rearrangement was associated with the T-R transition of the molecule.Crystal structure...

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“…In these experiments, the sample is exposed to the cw laser for various times, suddenly turned off, and the kinetics of CO rebinding to unliganded subunits is measured by optical absorption. Because the gel traps partially liganded T for many minutes, all of the tetramers are in the T quaternary structure during the experiment, as shown previously (34). Fig.…”

Section: Resultsmentioning

confidence: 54%

“…Confirmation of Tertiary r in Liganded Quaternary T. As a control and to confirm our interpretation of r tertiary in T quaternary in the pulsed laser photolysis experiments of Viappiani et al (34) (Fig. 2), we first carried out cw photolysis experiments on the T quaternary structure in the absence of allosteric effectors (T − ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In a previous study, we carried out nanosecond pulsed laser photolysis experiments on HbCO encapsulated and thereby trapped in either the T or R quaternary structures, corresponding to the arrangement of the four subunits in fully unliganded and fully liganded hemoglobin, respectively (Fig. 2B) (34). We discovered that, although the R and T quaternary structures show a single fast and slow CO rebinding phase in the presence of allosteric effectors, respectively, in the absence of allosteric effectors the T Significance Although the theoretical model of Monod, Wyman, and Changeux (MWC) is one of the most influential and highly cited theoretical models in bioscience, it fails to explain allosteric effects in hemoglobin, the paradigm of allostery, because their model considers only quaternary preequilibria.…”

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confidence: 99%

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Experimental basis for a new allosteric model for multisubunit proteins

Viappiani

Abbruzzetti

Ronda³

et al. 2014

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

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Monod, Wyman, and Changeux (MWC) explained allostery in multisubunit proteins with a widely applied theoretical model in which binding of small molecules, so-called allosteric effectors, affects reactivity by altering the equilibrium between more reactive (R) and less reactive (T) quaternary structures. In their model, each quaternary structure has a single reactivity. Here, we use silica gels to trap protein conformations and a new kind of laser photolysis experiment to show that hemoglobin, the paradigm of allostery, exhibits two ligand binding phases with the same fast and slow rates in both R and T quaternary structures. Allosteric effectors change the fraction of each phase but not the rates. These surprising results are readily explained by the simplest possible extension of the MWC model to include a preequilibrium between two tertiary conformations that have the same functional properties within each quaternary structure. They also have important implications for the long-standing question of a structural explanation for the difference in hemoglobin oxygen affinity of the two quaternary structures.S mall molecules can regulate protein reactivity by binding to residues distant from the active site, a phenomenon known as allostery. The classic theoretical model proposed by Monod, Wyman, and Changeux (MWC) (1, 2) for explaining this phenomenon considered only proteins with multiple subunits, and the two-state allosteric model of MWC was originally used by them to explain the functional properties of the hemoglobin tetramer, the "honorary enzyme" (1, 3). This famous model has since been applied to a wide variety of other systems in biology, including ligand-gated ion channels, G-protein-coupled receptors, nuclear receptors, and supramolecular assemblies such as chaperonins (4-7). In the case of hemoglobin, the paradigm of allostery, MWC makes two key postulates that have been extensively tested by experiments. Oxygen binding to the deoxy quaternary structure (T) is noncooperative; cooperativity arises from a shift in the population from the low-affinity T quaternary structure to the high-affinity R quaternary structure as the oxygen concentration is increased. The second postulate is that allosteric effectors regulate oxygen affinity by altering only the R ⇄ T preequilibrium. Investigations of hemoglobin focused primarily on the first postulate, which was finally confirmed by single-crystal oxygen-binding measurements that ruled out a sequential model (8) and ended a 25-y controversy (9-12). The second is of more general interest because it applies to all multisubunit proteins exhibiting allosteric behavior and has been known for many years to be inconsistent with the fact that allosteric effectors markedly affect oxygen affinity without changing the quaternary preequilibrium [see data summaries by Imai, Yonetani, and coworkers (13,14)]. The change in oxygen affinity constants, K T and K R , with conditions was interpreted as indicating that tertiary conformations must be affected or that more than two quat...

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

Cited by 108 publications

References 42 publications

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

A quantum-chemical picture of hemoglobin affinity

Experimental basis for a new allosteric model for multisubunit proteins

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