Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models.
Ontology highlight
ABSTRACT:
SUBMITTER: Henry ER
PROVIDER: S-EPMC4576146 | biostudies-literature | 2015 Sep
REPOSITORIES: biostudies-literature
Publications
Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models.
Henry Eric R ER Mozzarelli Andrea A Viappiani Cristiano C Abbruzzetti Stefania S Bettati Stefano S Ronda Luca L Bruno Stefano S Eaton William A WA
Biophysical journal 20150530 6
Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include ...[more]
Similar Datasets
Project description: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 pre-equilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.
| S-EPMC521967 | biostudies-literature
Project description:The canonical Wnt signaling pathway, mediated by ?-catenin, is crucially involved in development, adult stem cell tissue maintenance, and a host of diseases including cancer. We analyze existing mathematical models of Wnt and compare them to a new Wnt signaling model that targets spatial localization; our aim is to distinguish between the models and distill biological insight from them. Using Bayesian methods we infer parameters for each model from mammalian Wnt signaling data and find that all models can fit this time course. We appeal to algebraic methods (concepts from chemical reaction network theory and matroid theory) to analyze the models without recourse to specific parameter values. These approaches provide insight into aspects of Wnt regulation: the new model, via control of shuttling and degradation parameters, permits multiple stable steady states corresponding to stem-like vs. committed cell states in the differentiation hierarchy. Our analysis also identifies groups of variables that should be measured to fully characterize and discriminate between competing models, and thus serves as a guide for performing minimal experiments for model comparison.
| S-EPMC4352827 | biostudies-other
Project description:Deformation mechanisms in bcc metals, especially in dynamic regimes, show unusual complexity, which complicates their use in high-reliability applications. Here, we employ novel, high-velocity cylinder impact experiments to explore plastic anisotropy in single crystal specimens under high-rate loading. The bcc tantalum single crystals exhibit unusually high deformation localization and strong plastic anisotropy when compared to polycrystalline samples. Several impact orientations - [100], [110], [111] and [[Formula: see text]] - are characterized over a range of impact velocities to examine orientation-dependent mechanical behavior versus strain rate. Moreover, the anisotropy and localized plastic strain seen in the recovered cylinders exhibit strong axial symmetries which differed according to lattice orientation. Two-, three-, and four-fold symmetries are observed. We propose a simple crystallographic argument, based on the Schmid law, to understand the observed symmetries. These tests are the first to explore the role of single-crystal orientation in Taylor impact tests and they clearly demonstrate the importance of crystallography in high strain rate and temperature deformation regimes. These results provide critical data to allow dramatically improved high-rate crystal plasticity models and will spur renewed interest in the role of crystallography to deformation in dynamics regimes.
| S-EPMC5883051 | biostudies-literature
Project description:The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets--called hidden allosteric sites--remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein's structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target--TEM-1 ?-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.
| S-EPMC4352775 | biostudies-literature
Project description:The mechanical behavior of single crystalline, micro-sized copper is investigated in the context of cantilever beam bending experiments. Particular focus is on the role of geometrically necessary dislocations (GNDs) during bending-dominated load conditions and their impact on the characteristic bending size effect. Three different sample sizes are considered in this work with main variation in thickness. A gradient extended crystal plasticity model is presented and applied in a three-dimensional finite-element (FE) framework considering slip system-based edge and screw components of the dislocation density vector. The underlying mathematical model contains non-standard evolution equations for GNDs, crystal-specific interaction relations, and higher-order boundary conditions. Moreover, two element formulations are examined and compared with respect to size-independent as well as size-dependent bending behavior. The first formulation is based on a linear interpolation of the displacement and the GND density field together with a full integration scheme whereas the second is based on a mixed interpolation scheme. While the GND density fields are treated equivalently, the displacement field is interpolated quadratically in combination with a reduced integration scheme. Computational results indicate that GND storage in small cantilever beams strongly influences the evolution of statistically stored dislocations (SSDs) and, hence, the distribution of the total dislocation density. As a particular example, the mechanical bending behavior in the case of a physically motivated limitation of GND storage is studied. The resulting impact on the mechanical bending response as well as on the predicted size effect is analyzed. Obtained results are discussed and related to experimental findings from the literature.
| S-EPMC5503410 | biostudies-other
Project description:Room temperature biospecimen storage for prolonged periods is essential to eliminate energy consumption by ultra-low freezing or refrigeration-based storage techniques. State of the art practices that sufficiently minimize the direct or hidden costs associated with cold-chain logistics include ambient temperature storage of biospecimens (i.e., DNA, RNA, proteins, lipids) in the dry state. However, the biospecimens are still well-exposed to the stress associated with drying and reconstitution cycles, which augments the pre-analytical degradation of biospecimens prior to their downstream processing. An aqueous storage solution that can eliminate these stresses which are correlated to several cycles of drying/rehydration or freezing of biospecimens, is yet to be achieved by any current technology. In our study, we have addressed this room temperature biospecimen-protection challenge using aqueous capture and release gels for optimized storage (Bio-CaRGOS) of biospecimens. Herein, we have demonstrated a single-step ∼95% recovery of a metalloprotein hemoglobin at room temperature using a cost-effective standard microwave-based aqueous formulation of Bio-CaRGOS. Although hemoglobin samples are currently stored at sub-zero or under refrigeration (4 °C) conditions to avoid loss of integrity and an unpredictable diagnosis during their downstream assays, our results have displayed an unprecedented room temperature integrity preservation of hemoglobin. Bio-CaRGOS formulations efficiently preserve hemoglobin in its native state, with single-step protein recovery of ∼95% at ambient conditions (1 month) and ∼96% (7 months) under refrigeration conditions. In contrast, two-thirds of the control samples degrade under ambient (1 month) and refrigeration (7 months) settings.
| S-EPMC8697545 | biostudies-literature
Project description:Hemoglobin is the prototypic allosteric protein. Still, its molecular allosteric mechanism is not fully understood. To elucidate the mechanism of cooperativity on an atomistic level, we developed a novel computational technique to analyse the coupling of tertiary and quaternary motions. From Molecular Dynamics simulations showing spontaneous quaternary transitions, we separated the transition trajectories into two orthogonal sets of motions: one consisting of intra-chain motions only (referred to as tertiary-only) and one consisting of global inter-chain motions only (referred to as quaternary-only). The two underlying subspaces are orthogonal by construction and their direct sum is the space of full motions. Using Functional Mode Analysis, we were able to identify a collective coordinate within the tertiary-only subspace that is correlated to the most dominant motion within the quaternary-only motions, hence providing direct insight into the allosteric coupling mechanism between tertiary and quaternary conformation changes. This coupling-motion is substantially different from tertiary structure changes between the crystallographic structures of the T- and R-state. We found that hemoglobin's allosteric mechanism of communication between subunits is equally based on hydrogen bonds and steric interactions. In addition, we were able to affect the T-to-R transition rates by choosing different histidine protonation states, thereby providing a possible atomistic explanation for the Bohr effect.
| S-EPMC3777908 | biostudies-literature
Project description:The serine protease ?-thrombin is a dual-action protein that mediates the blood-clotting cascade. Thrombin alone is a procoagulant, cleaving fibrinogen to make the fibrin clot, but the thrombin-thrombomodulin (TM) complex initiates the anticoagulant pathway by cleaving protein C. A TM fragment consisting of only the fifth and sixth EGF-like domains (TM56) is sufficient to bind thrombin, but the presence of the fourth EGF-like domain (TM456) is critical to induce the anticoagulant activity of thrombin. Crystallography of the thrombin-TM456 complex revealed no significant structural changes in thrombin, suggesting that TM4 may only provide a scaffold for optimal alignment of protein C for its cleavage by thrombin. However, a variety of experimental data have suggested that the presence of TM4 may affect the dynamic properties of the active site loops. In the present work, we have used both conventional and accelerated molecular dynamics simulation to study the structural dynamic properties of thrombin, thrombin:TM56, and thrombin:TM456 across a broad range of time scales. Two distinct yet interrelated allosteric pathways are identified that mediate both the pro- and anticoagulant activities of thrombin. One allosteric pathway, which is present in both thrombin:TM56 and thrombin:TM456, directly links the TM5 domain to the thrombin active site. The other allosteric pathway, which is only present on slow time scales in the presence of the TM4 domain, involves an extended network of correlated motions linking the TM4 and TM5 domains and the active site loops of thrombin.
| S-EPMC3535651 | biostudies-literature
Project description:Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide-angle X-ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.
| S-EPMC5326564 | biostudies-literature
Project description:Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726-734], the subunit motions do not follow the textbook description of a simple rotation of one ??-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 ?s) as well as a slow (20 ?s) component and provide a testable model for single molecule FRET experiments.
| S-EPMC3078355 | biostudies-literature