Intermediate Metastable State Research Articles

Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation.

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.

Conformational transitions of bio-molecular systems studied using adaptive bond bending elastic network model

Conformational transitions in proteins are essential for biological functions. Such transitions involve short-lived conformational states that are difficult to detect experimentally. Motivated by the utility and efficiency of elastic network models for describing the collective dynamics of proteins, in this study we present a new adaptive bond bending elastic network model for studying the conformational transition pathway of adenylate kinase and leucine transporter. Assuming that all the native contacts within these proteins are identical, we assigned a bond binding energy to the native contacts and computed the free energy of each image along the transition pathway. After plotting the free energy profiles, we found a few metastable intermediate states, separated by the free energy barriers. The observed intermediate states and the transition pathways for all proteins were in good agreement with those of the other methods and molecular dynamics simulations.

Quantum Approach to Fast Protein-Folding Time**Supported by National Key R&D Program of China under Grant No 2017YFA0304304, and partially by the Fundamental Research Funds for the Central Universities.

In the traditional random-conformational-search model, various hypotheses with a series of meta-stable intermediate states were proposed to resolve the Levinthal paradox in protein-folding time. Here we introduce a quantum strategy to formulate protein folding as a quantum walk on a definite graph, which provides us a general framework without making hypotheses. Evaluating it by the mean of first passage time, we find that the folding time via our quantum approach is much shorter than the one obtained via classical random walks. This idea is expected to evoke more insights for future studies.

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ∼1 ms, while all other receptors are thought to become activated within ∼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 ∼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 ∼ 20 ms). Receptor deactivation occurred with time constants of ∼40 and ∼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.

Influence of Y–Gd ratio on phase formation and spectroscopic properties of NaGd0.8−xYxYb0.17Er0.03F4 solid solutions

The work is devoted to the study of phase formation in the NaGd0.8Yb0.17Er0.03F4–NaY0.8Yb0.17Er0.03F4 system. The effect of the Gd3+ и Y3+ ions concentrations ratio on the phase composition and lattice parameters of the resulting solid solutions, the spectral-luminescent properties, and the heating temperature under laser irradiation were studied. It is shown that an increase of the molar fraction of Gd3+ ions in the composition leads to the NaGdF4 hexagonal phase formation and to increase in the lattice parameter values. The measured upconversion efficiency for NaGd0.8Yb0.17Er0.03F4 is significantly lower than for NaY0.8Yb0.17Er0.03F4. In order to interpret the obtained result, a theoretical model on the basis of rate equations system was used, taking into account the experimentally determined lattice parameters. It was shown that as the lattice parameters increase, the distance between the active ions increases and the probability of energy transfer decreases. Shorter lifetimes of the excited states for NaGdF4 cause an increase in the back energy transfer probability. Additionally, the shorter lifetime of intermediate metastable states in the NaGdF4 matrix negatively affects the efficiency of the upconversion processes. All these processes result in emitting states populations decrease and a decrease in upconversion efficiency, respectively. According to the calculations, the efficiency for NaGd0.8Yb0.17Er0.03F4 is reduced by 40%. Experimentally observed decrease was much higher. We propose that the additional decrease is due to the influence of factors not considered in the model. The size reduction for NaGdF4 particles was observed (350 nm in comparison to 1 µm for NaYF4 particles). The second additional factor, which leads to experimental efficiency decrease in comparison with theoretical value, is the heating. The study of the samples heating temperature under laser radiation showed that the increase in the gadolinium ions fraction in the composition results in the sample heating temperature increase. The NaGd0.8Yb0.17Er0.03F4 sample was heated by laser radiation by 24.7 °C, while the sample NaY0.8Yb0.17Er0.03F4 was heated by 5.6 °C.

Revealing the role of lattice distortions in the hydrogen-induced metal-insulator transition of SmNiO3

The discovery of hydrogen-induced electronic phase transitions in strongly correlated materials such as rare-earth nickelates has opened up a new paradigm in regulating materials’ properties for both fundamental study and technological applications. However, the microscopic understanding of how protons and electrons behave in the phase transition is lacking, mainly due to the difficulty in the characterization of the hydrogen doping level. Here, we demonstrate the quantification and trajectory of hydrogen in strain-regulated SmNiO3 by using nuclear reaction analysis. Introducing 2.4% of elastic strain in SmNiO3 reduces the incorporated hydrogen concentration from ~1021 cm−3 to ~1020 cm−3. Unexpectedly, despite a lower hydrogen concentration, a more significant modification in resistivity is observed for tensile-strained SmNiO3, substantially different from the previous understanding. We argue that this transition is explained by an intermediate metastable state occurring in the transient diffusion process of hydrogen, despite the absence of hydrogen at the post-transition stage.

Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation

Peptidoglycan (PG) is the main constituent of the bacterial cell wall. The enzyme UDP‑N‑acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridinediphospho‑N‑acetylglucosamine, which is the first committed step of PG biosynthesis. In this study, we have systematically examined the urea-induced unfolding of Acinetobacter baumannii MurA (AbMurA) using various optical spectroscopic techniques and molecular dynamics (MD) simulations. The urea-induced unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated between 3.0 and 4.0 M. Above 6.0 M urea, AbMurA gets completely unfolded. The transition from the native structure to the partially unfolded metastable state involves ~30% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes AbMurA structure weakening hydrophobic interactions and the hydrogen bond network. We observed a clear correlation between both in vitro and in silico studies. To our knowledge, this is also the first report on unfolding/stability analysis of any MurA enzyme.

General framework for nonclassical nucleation

A great deal of experimental evidence suggests that a wide spectrum of phase transitions occur in a multistage manner via the appearance and subsequent transformation of intermediate metastable states. Such multistage mechanisms cannot be explained within the realm of the classical nucleation framework. Hence, there is a strong need to develop new theoretical tools to explain the occurrence and nature of these ubiquitous intermediate phases. Here we outline a unified and self-consistent theoretical framework to describe both classical and nonclassical nucleation. Our framework provides a detailed explanation of the whole multistage nucleation pathway showing in particular that the pathway involves a single energy barrier and it passes through a dense phase, starting from a low-density initial phase, before reaching the final stable state. Moreover, we demonstrate that the kinetics of matter inside subcritical clusters favors the formation of nucleation clusters with an intermediate density, i.e. nucleation precursors. Remarkably, these nucleation precursors are not associated with a local minimum of the thermodynamic potential, as commonly assumed in previous phenomenological approaches. On the contrary, we find that they emerge due to the competition between thermodynamics and kinetics of cluster formation. Thus, the mechanism uncovered for the formation of intermediate phases can be used to explain recently reported experimental findings in crystallization: up to now such phases were assumed a consequence of some complex energy landscape with multiple energy minima. Using fundamental concepts from kinetics and thermodynamics, we provide a satisfactory explanation for the so-called nonclassical nucleation pathways observed in experiments.

Direct observation of pure pentavalent uranium in U2O5 thin films by high resolution photoemission spectroscopy

Thin films of the elusive intermediate uranium oxide U2O5 have been prepared by exposing UO3 precursor multilayers to atomic hydrogen. Electron photoemission spectra measured about the uranium 4f core-level doublet contain sharp satellites separated by 7.9(1) eV from the 4f main lines, whilst satellites characteristics of the U(IV) and U(VI) oxidation states, expected respectively at 6.9(1) and 9.7(1) eV from the main 4f lines, are absent. This shows that uranium ions in the films are in a pure pentavalent oxidation state, in contrast to previous investigations of binary oxides claiming that U(V) occurs only as a metastable intermediate state coexisting with U(IV) and U(VI) species. The ratio between the 5f valence band and 4f core-level uranium photoemission intensities decreases by about 50% from UO2 to U2O5, which is consistent with the 5f 2 (UO2) and 5f 1 (U2O5) electronic configurations of the initial state. Our studies conclusively establish the stability of uranium pentoxide.

In situ X-ray scattering observation of two-dimensional interfacial colloidal crystallization

Charged colloids at interfaces hold such a simple configuration that their interactions are supposed to be fully elucidated in the framework of classical electrostatics, yet the mysterious existence of attractive forces between these like-charged particles has puzzled the scientific community for decades. Here, we perform the in situ grazing-incidence small-angle X-ray scattering study of the dynamic self-assembling process of two-dimensional interfacial colloids. This approach allows simultaneous monitoring of the in-plane structure and ordering and the out-of-plane immersion depth variation. Upon compression, the system undergoes multiple metastable intermediate states before the stable hexagonal close-packed monolayer forms under van der Waals attraction. Remarkably, the immersion depth of colloidal particles is found to increase as the interparticle distance decreases. Numerical simulations demonstrate the interface around a colloid is deformed by the electrostatic force from its neighboring particles, which induces the long-range capillary attraction.

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest. In this study, we have investigated the biophysical behavior of intermediate ensembles employing all-atom molecular dynamics simulations in 8 M urea accelerated with high temperatures to achieve unfolded states in a confined computation time. The cumulative results of the 2.75 μs simulations show that unfolding of the NTD at 350 K evolves through different stable and meta-stable intermediate states. The free-energy landscape reveals two meta-stable intermediates (IN and IU) stabilized by non-native interactions, which are largely hydrophilic and highly energetically frustrated. A single buried tryptophan residue, W80, undergoes solvent exposure to different extents during unfolding; this suggests a structurally heterogeneous population of intermediate ensembles. Furthermore, the structure properties of the IN state show a resemblance to the molten globule (MG) state with most of the secondary structures intact. The unfolding of the NTD is initiated by the loss of β-strands, and the unfolded (U) states exhibit a population of non-native α-helices. These non-native unfolded intermediate ensembles may mediate protein oligomerization, leading to the formation of pathological, irreversible aggregates, characteristics of disease pathogenesis.

Metastable State during Melting and Solid-Solid Phase Transition of [CnMim][NO3] (n = 4-12) Ionic Liquids by Molecular Dynamics Simulation.

We simulate the heating process of ionic liquids [CnMim][NO3] (n = 4, 6, 8, 10, 12), abbreviated as Cn, by means of molecular dynamics (MD) simulation starting from a manually constructed triclinic crystal structure composed of polar layers containing anions and cationic head groups and nonpolar regions in between containing cationic alkyl side chains. During the heating process starting from 200 K, each system undergoes first a solid-solid phase transition at a lower temperature, and then a melting phase transition at a higher temperature to an isotropic liquid state (C4, C6, and C8) or to a liquid crystal state (C10 and C12). After the solid-solid phase transition, all systems keep the triclinic space symmetry, but have a different set of lattice constants. C4 has a more significant structural change in the nonpolar regions which narrows the layer spacing, while the layer spacings of other systems change little, which can be qualitatively understood by considering that the contribution of the effective van der Waals interaction in the nonpolar regions (abbreviated as EF1) to free energy becomes stronger with increasing side-chain length, and at the same time the contribution of the effective electrostatic interaction in the polar layers (abbreviated as EF2) to free energy remains almost the same. The melting phase transitions of all systems except C6 are found to be a two-step process with an intermediate metastable state appeared during the melting from the crystal state to the liquid or liquid crystal state. Because the contribution of EF2 to the free energy is larger than EF1, the metastable state of C4 has the feature of having higher ordered polar layers and lower ordered side-chain orientation. By contrast, C8-C12 have the feature of having lower ordered polar layers and higher ordered side-chain orientation, because for these systems, the contribution of EF2 to the free energy is smaller than EF1. No metastable state is found for C6 because the free-energy contribution of EF1 is balanced with EF2.

Studying Kv Channels Function using Computational Methods.

In recent years, molecular modeling techniques, combined with MD simulations, provided significant insights on voltage-gated (Kv) potassium channels intrinsic properties. Among the success stories are the highlight of molecular level details of the effects of mutations, the unraveling of several metastable intermediate states, and the influence of a particular lipid, PIP2, in the stability and the modulation of Kv channel function. These computational studies offered a detailed view that could not have been reached through experimental studies alone. With the increase of cross disciplinary studies, numerous experiments provided validation of these computational results, which endows an increase in the reliability of molecular modeling for the study of Kv channels. This chapter offers a description of the main techniques used to model Kv channels at the atomistic level.

Allocating dissipation across a molecular machine cycle to maximize flux

Biomolecular machines consume free energy to break symmetry and make directed progress. Nonequilibrium ATP concentrations are the typical free energy source, with one cycle of a molecular machine consuming a certain number of ATP, providing a fixed free energy budget. Since evolution is expected to favor rapid-turnover machines that operate efficiently, we investigate how this free energy budget can be allocated to maximize flux. Unconstrained optimization eliminates intermediate metastable states, indicating that flux is enhanced in molecular machines with fewer states. When maintaining a set number of states, we show that-in contrast to previous findings-the flux-maximizing allocation of dissipation is not even. This result is consistent with the coexistence of both "irreversible" and reversible transitions in molecular machine models that successfully describe experimental data, which suggests that, in evolved machines, different transitions differ significantly in their dissipation.

Generalized parametric model for phase transitions in the presence of an intermediate metastable state and its application

The previously proposed model for the kinetics of first-order phase transitions (Barsuk et al., 2013) is generalized for r order and m control parameters. Bifurcation and stability analyses of the equilibrium states in thermodynamic systems described by the Landau-type kinetic potential with two order parameters is performed both in the absence of an external field, and in the presence of constant and periodic external fields. Kinetics of thermodynamic systems described by such potential in a small neighborhood of the equilibrium states is also studied. Mean transition time for lysozyme protein in dependence of control parameters is obtained based on the developed model. A detailed bifurcation analysis of the cubic equation solutions is given in Appendix.

Enhanced readout of spin states in double quantum dot

We investigate a spin-to-charge conversion mechanism which maps the spin singlet and triplet states to two charge states differing by one electron mediated by an intermediate metastable charge state. This mechanism allows us to observe fringes in the spin-unblocked region beyond the triplet transition line in the measurement of the exchange oscillations between singlet and triplet states in a four-electron double quantum dot. Moreover, these fringes are amplified and π-phase shifted, compared with those in the spin blockade region. Unlike the signal enhancement mechanism reported before which produces similar effects, this mechanism only requires one dot coupling to the lead, which is a commonly encountered case especially in imperfect devices. Besides, the crucial tunnel rate asymmetry is provided by the dependence on spin state, not by the asymmetric couplings to the leads. We also design a scheme to control the amplification process, which enables us to extract the relevant time parameters. This mechanism will have potential applications in future investigations of spin qubits.

De Sitter Space as a Resonance.

A quantum mechanical formulation of de Sitter cosmological spacetimes still eludes string theory. In this Letter we conjecture a potentially rigorous framework in which the status of de Sitter space is the same as that of a resonance in a scattering process. We conjecture that transition amplitudes between certain states with asymptotically supersymmetric flat vacua contain resonant pole characteristic metastable intermediate states. A calculation employing constrained instantons illustrates this idea.

De Sitter harmonies: Cosmological spacetimes as resonances

The aim of this work is to provided the details of a calculation summarized in the recent paper by Maltz and Susskind which conjectured a potentially rigorous framework where the status of de Sitter space is the same as that of a resonance in a scattering process. The conjecture being that transition amplitudes between certain states with asymptotically supersymmetric flat vacua contain resonant poles characteristic metastable intermediate states. A calculation employing constrained instantons is presented that illustrates this idea.

Metastable states in terminal orientation of hinged symmetric bodies in a flow

Symmetric bodies such as cylinders and spheroidal bodies, in their terminal stable states, are long known to have their long axis align themselves perpendicular to the direction of the flow. This property has been confirmed in primarily sedimentation based theoretical, experimental and numerical techniques and the transition to a terminal stable state is believed to coincide with the onset of significant inertial effects in the flow. However, the threshold at which this transition occurs is yet unknown. We conduct modified experiments with hinged bodies and a CFD study to examine the nature of the transition of prolate spheroids and cylinders of various aspect ratios, from initial to their terminal stable equilibrium. Our experiments and numerics, both reveal the presence of intermediate metastable states which are sensitive to the flow Reynolds number and physical attributes of the immersed body and gradually lead to the stable state.

ChemInform Abstract: Recent Developments in Biological Water Oxidation

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