Intermediate Metastable State Research Articles

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Vliyanie shuma na rezistivnoe pereklyuchenie memristora na osnove stabilizirovannogo dioksida tsirkoniya

The effect of Gaussian noise on the switching of a ZrO2(Y) based memristor from the low resistance state (LRS) into the high resistance state (HRS) including transitions from the LRS into intermediate metastable states has been studied. The series of positive (with addition of the noise signal or without the one) and negative rectangular voltage pulses were used as the switching signals. The adding of noise to the switching signal initiated the switching of the memristor from the LRS into the HRS at smaller pulse magnitudes than in the case of switching by the rectangular pulses without adding the noise. A necessary (preset) HRS can be achieved passing the intermediate states by adding the noise with certain parameters to the rectangular switching pulses. The resistive switching is performed without application of adaptive switching protocols. The results of the present study can be applied in the development of innovative memristor switching protocols.

Modeling the Effect on a Novel Fungal Peptaibol Placed in an All-Atom Bacterial Membrane Mimicking System via Accelerated Molecular Dynamics Simulations.

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

A Travel through Landscapes of Seed Dormancy

Basic features of seed dormancy are illustrated. The seed overall regulatory network governs seed metabolism and development, and it is coordinated by plant hormones. A functional model focused on abscisic acid (ABA), the foremost plant hormone in dormancy, is used as a framework to critically discuss the literature. Gibberellins (GAs) have a main role in germination, and the ABA–GAs balance is a typical feature of the seed state: ABA dominates during dormancy and GAs prevail through germination. Thus, the literature converges toward envisaging the development switch between dormancy and germination as represented by the ABA/GAs ratio. The ABA–GAs antagonism is based on mutual inhibition, a feature of the regulatory network architecture that characterizes development trajectories based on a regulatory circuit with a bistable switch. Properties of such kind of regulatory architecture are introduced step by step, and it is shown that seed development—toward either dormancy or germination—is more properly represented by a tristable regulatory circuit, whose intermediate metastable states ultimately take one or the other development trajectory. Although the ABA/GAs ratio can conveniently represent the state of the seed overall regulatory network along the seed development trajectory, specific (unknown) dormancy factors are required to determine the development trajectory. The development landscape is shown to provide a well-suited representation of seed states travelling along developmental trajectories, particularly when the states are envisioned as regulatory circuits. Looking at seed dormancy in terms of regulatory circuits and development landscapes offers a valuable perspective to improve our understanding of this biological phenomenon.

Reentrant canonical spin-glass dynamics and tunable field-induced transitions in (GeMn)Co2O4 Kagomé lattice

We report on the reentrant canonical semi spin-glass characteristics and controllable field-induced transitions in distorted Kagomé symmetry of (GeMn)Co2O4. This B-site spinel exhibits complicated, yet interesting magnetic behaviour in which the longitudinal ferrimagnetic (FiM) order sets in below the Néel temperature T FN ∼ 77 K due to uneven moments of divalent Co (↑ 5.33 μ B) and tetravalent Mn (↓ 3.87 μ B) which coexists with transverse spin-glass state below 72.85 K. Such complicated magnetic behaviour is suggested to result from the competing anisotropic superexchange interactions (J AB/k B ∼ 4.3 K, J AA/k B ∼ −6.2 K and J BB/k B ∼ −3.3 K) between the cations, which is extracted following the Néel’s expression for the two-sublattice model of FiM. Dynamical susceptibility (χ ac (f, T)) and relaxation of thermoremanent magnetization, M TRM (t) data have been analysed by means of the empirical scaling-laws such as Vogel–Fulcher law and Power law of critical slowing down. Both of which reveal the reentrant spin-glass like character which evolves through a number of intermediate metastable states. The magnitude of Mydosh parameter (Ω ∼ 0.002), critical exponent zυ = (6.7 ± 0.07), spin relaxation time τ 0 = (2.33 ± 0.1) × 10−18 s, activation energy E a/k B = (69.8 ± 0.95) K and interparticle interaction strength (T 0 = 71.6 K) provide the experimental evidences for canonical spin-glass state below the spin freezing temperature T F = 72.85 K. The field dependence of T F obtained from χ ac (T) follows the irreversibility in terms of de Almeida–Thouless mean-field instability in which the magnitude of crossover scaling exponent Φ turns out to be ∼2.9 for the (Ge0.8Mn0.2)Co2O4. Isothermal magnetization plots reveal two field-induced transitions across 9.52 kOe (H SF1) and 45.6 kOe (H SF2) associated with the FiM domains and spin-flip transition, respectively. Analysis of the inverse paramagnetic susceptibility after subtracting the temperature independent diamagnetic term (=−3 × 10−3 emu mol−1 Oe−1) results in the effective magnetic moment = 7.654 μ B/f.u. This agrees well with the theoretically obtained = 7.58 μ B/f.u. resulting the cation distribution in support of the Hund’s ground state spin configuration and of Mn4+ and Co2+, respectively. The H–T phase diagram has been established by analysing all the parameters (T F(H), T FN(H), H SF1(T) and H SF2(T)) extracted from various magnetization measurements. This diagram enables clear differentiation among the different phases of the (GeMn)Co2O4 and also illustrates the demarcation between short-range and long-range ordered regions.

Electrically induced transformation of cholesteric droplets under homeotropic boundary conditions

Cholesteric droplets under homeotropic boundary conditions demonstrate a rich variety of possible orientational structures. We have implemented electrically controlled switching of such droplets between two classes of stable states: i) structures with cylindrical cholesteric layers and ii) layer-like structures with one or more λ+1/2-disclinations. Structure relaxation after switching off the voltage proceeded via the fast (less than 1 s), the slow (more than 1 s), and the stabilization stages (∼10 hours). We investigated intermediate long-lived metastable states occurring in the relaxation process and explained the formation of various structures in cholesteric droplets. The fast and the slow stages of relaxation were found to be sensitive to the voltage reduction regime. We have determined the combinations of droplet size and voltage reduction regimes leading to the two types of structures after relaxation.

Distinct activation mechanisms regulate subtype selectivity of Cannabinoid receptors

Design of cannabinergic subtype selective ligands is challenging because of high sequence and structural similarities of cannabinoid receptors (CB1 and CB2). We hypothesize that the subtype selectivity of designed selective ligands can be explained by the ligand binding to the conformationally distinct states between cannabinoid receptors. Analysis of ~ 700 μs of unbiased simulations using Markov state models and VAMPnets identifies the similarities and distinctions between the activation mechanism of both receptors. Structural and dynamic comparisons of metastable intermediate states allow us to observe the distinction in the binding pocket volume change during CB1 and CB2 activation. Docking analysis reveals that only a few of the intermediate metastable states of CB1 show high affinity towards CB2 selective agonists. In contrast, all the CB2 metastable states show a similar affinity for these agonists. These results mechanistically explain the subtype selectivity of these agonists by deciphering the activation mechanism of cannabinoid receptors.

Wnt signaling and the regulation of pluripotency.

The role of Wnt signaling in stem cells has been mired in seemingly contradictory findings. On one hand, Wnt has been heralded as a self-renewal factor. On the other hand, Wnt's association with differentiation and lineage commitment is indisputable. This apparent contradiction is particularly evident in pluripotent stem cells, where Wnt promotes self-renewal as well as differentiation. To resolve this discrepancy one must delve into fundamental principles of pluripotency and gain an appreciation for the concept of pluripotency states, which exist in a continuum with intermediate metastable states, some of which have been stabilized in vitro. Wnt signaling is a critical regulator of transitions between pluripotent states. Here, we will discuss Wnt's roles in maintaining pluripotency, promoting differentiation, as well as stimulating reprogramming of somatic cells to an induced pluripotent state.

How a single mutation alters the protein structure: a simulation investigation on protein tyrosine phosphatase SHP2.

Protein tyrosine phosphatase SHP2 is a key regulator modulating several signaling pathways. The oncogenic mutation E76K in SHP2 releases the enzyme from an autoinhibited, closed conformation into an active, open conformation. Here, we investigated the conformational dynamics of SHP2 and the effect of the E76K mutation on its conformational ensemble via extensive molecular dynamics (MD) and metadynamics (MetaD) simulations. Our simulations provide atomistic details on how the E76K mutated SHP2 prefers the open state and also reveal that the transition between the closed and the open states is highly collective. Several intermediate metastable states during the conformational transition between the closed and the open states were also investigated. Understanding how the single E76K mutation induces the conformational change in SHP2 could facilitate the further design of SHP2 inhibitors.

On-Surface Synthesis and Evolution of Self-Assembled Poly(p-phenylene) Chains on Ag(111): A Joint Experimental and Theoretical Study.

The growth of controlled 1D carbon-based nanostructures on metal surfaces is a multistep process whose path, activation energies, and intermediate metastable states strongly depend on the employed substrate. Whereas this process has been extensively studied on gold, less work has been dedicated to silver surfaces, which have a rather different catalytic activity. In this work, we present an experimental and theoretical investigation of the growth of poly-p-phenylene (PPP) chains and subsequent narrow graphene ribbons starting from 4,4″-dibromo-p-terphenyl molecular precursors deposited at the silver surface. By combing scanning tunneling microscopy (STM) imaging and density functional theory (DFT) simulations, we describe the molecular morphology and organization at different steps of the growth process and we discuss the stability and conversion of the encountered species on the basis of calculated thermodynamic quantities. Unlike the case of gold, at the debromination step we observe the appearance of organometallic molecules and chains, which can be explained by their negative formation energy in the presence of a silver adatom reservoir. At the dehydrogenation temperature, the persistence of intercalated Br atoms hinders the formation of well-structured graphene ribbons, which are instead observed on gold, leading only to a partial lateral coupling of the PPP chains. We numerically derive very different activation energies for Br desorption from the Ag and Au surfaces, thereby confirming the importance of this process in defining the kinetics of the formation of molecular chains and graphene ribbons on different metal surfaces.

Metal-Ion-Induced Evolution of Phenylalanine Self-Assembly: Structural Polymorphism of Novel Metastable Intermediates.

The self-assembly of aromatic amino acids has been widely studied due to their ability to form well-defined amyloid-like fibrillar structures. Herein, for the first time, we report the existence of different metastable intermediate states of diverse morphologies, for example, droplets, spheres, vesicles, flowers, and toroids, that are sequentially formed in aqueous medium during the self-assembly process of phenylalanine in the presence of different divalent (Zn2+, Cd2+, and Hg2+) and trivalent (Al3+, Ga3+, and In3+) metal ions having low pKa values. Due to metal ion-amino acid coordination and strong hydrophobic interaction induced by these metal ions, spherical aggregates are obtained at the initial stage of the structural evolution and further transformed into other intermediate states. Our work may facilitate understanding of the role of metal ions in the amino acid self-assembly process and broaden future applications of the obtained nanostructures in drug delivery, tissue engineering, bioimaging, biocatalysis, and other fields.

Concerted versus stepwise mechanisms of cyclic proton transfer: Experiments, simulations, and current challenges

Proton transfer (PT) is a process of fundamental importance in hydrogen (H)-bonded systems. At cryogenic or moderate temperatures, pronounced quantum tunneling may happen due to the light mass of H. Single PT processes have been extensively studied. However, for PT involving multiple protons, our understanding remains in its infancy stage due to the complicated interplay between the high-dimensional nature of the process and the quantum nature of tunneling. Cyclic H-bonded systems are typical examples of this, where PT can happen separately via a “stepwise” mechanism or collectively via a “concerted” mechanism. In the first scenario, some protons hop first, typically resulting in metastable intermediate states (ISs) and the reaction pathway passes through multiple transition states. Whilst in the concerted mechanism, all protons move simultaneously, resulting in only one barrier along the path. Here, we review previous experimental and theoretical studies probing quantum tunneling in several representative systems for cyclic PT, with more focus on recent theoretical findings with path-integral based methods. For gas-phase porphyrin and porphycene, as well as porphycene on a metal surface, theoretical predictions are consistent with experimental observations, and enhance our understanding of the processes. Yet, discrepancies in the PT kinetic isotope effects between experiment and theory appear in two systems, most noticeably in water tetramer adsorbed on NaCl (001) surface, and also hinted in porphycene adsorbed on Ag (110) surface. In ice I h, controversy surrounding concerted PT remains even between experiments. Despite of the recent progress in both theoretical methods and experimental techniques, multiple PT processes in cyclic H-bonded systems remain to be mysterious.

Spin-glass behavior in Li3NiCuBiO6: a two-dimensional distorted honeycomb-lattice

We report a detailed experimental study on the structural and magnetic properties of Li3NiCuBiO6 by means of various characterization techniques. It crystallizes into a monoclinic crystal structure composed of a layered magnetic honeycomb lattice along the c-axis. The existence of glassy state below 4 K is indicated by dc and ac susceptibility measurements. Magnetic contribution to the total heat capacity also peaks around the freezing temperature, and its linear temperature dependence backs our claim of a glassy state in the compound. The calculated magnetic entropy unveils that only ∼26% of the total entropy is released for the system (), and a tremendous amount of spin entropy is still retained in the system. Further, analysis of the frequency-dependent freezing temperature with the help of power law confirms the presence of a spin glass state. Moreover, the appearance of magnetic memory and relaxation effect below freezing temperature manifest the development of the system via a large number of intermediate metastable states. All these measurements confirm the spin-glass behavior of the compound. We consider the presence of different magnetic atoms in honeycomb lattice as the main driving factor for the spin-glass ground state.

New insights into the formation mechanism of zircon in a ZrO2–SiO2 nanocrystalline glass-ceramic: A TEM study

We have previously observed that doping of Ca ions was beneficial to the formation of zircon (ZrSiO4). It is well known that synthetic ZrSiO4 is typically formed via a solid-state reaction between ZrO2 and SiO2, in which the interfaces between the reactant and resultant play an important role. However, the interfaces are lacking detailed microstructural observation. This follow-up study aims at exploring the formation mechanism of ZrSiO4 by inspecting the interfaces at the nano and atomic scales with TEM techniques. Results demonstrated that ZrSiO4 was formed in the Ca-doped sample after sintering at 1200 °C, whereas, no ZrSiO4 was formed in the undoped sample even after sintering at 1230 °C. The Ca-doped sample consisted of a continuous ZrSiO4 matrix with dispersed ZrO2 nanocrystallites. Doping of Ca ions promoted the formation of ZrSiO4 by causing lattice distortion and oxygen vacancies in ZrO2 lattices. Thin amorphous grain boundary complexions were found between ZrO2 nanocrystallites and between ZrO2 and ZrSiO4 crystallites. These amorphous complexions acted as reaction sites and an intermediate metastable state for the solid-state reaction. A detailed formation mechanism of ZrSiO4 at the nanometer scale and atomic scale has been proposed.

Rydberg electromagnetically induced transparency and absorption of strontium triplet states in a weak microwave field

We study theoretically laser excitation of Rydberg triplet states of strontium atoms in the presence of weak microwave (MW) fields. Starting from the ground state $5s^2\,^1S_0$, the Rydberg excitation is realized through the metastable, triplet $5s5p\,^3P_1$ state, whose decay rate $\gamma_2$ is $2\pi\times 7.5$ kHz, much smaller than the one in the singlet state or alkali atoms. The influences of $\gamma_2$ on the transparency and absorption spectrum in the electromagnetically induced transparency (EIT), and electromagnetically induced absorption (EIA) regime are examined. Narrow transparent windows (EIT) or absorption peaks (EIA) are found, whose distance in the spectrum depends on the Rabi frequency of the weak MW field. It is found that the spectrum exhibits higher contrast than using the singlet state or alkali atoms in typical situations. Using the metastable intermediate state, we find that resonance fluorescence of Sr gases exhibits very narrow peaks, which are modulated by the MW field. When the MW field is weaker than the probe and control light, the spectrum distance of these peaks are linearly proportional to $\Omega_m$. This allows us to propose a new way to sense very weak MW fields through resonance fluorescence. Our study shows that the Sr triplet state could be used to develop the Rydberg MW electrometry that gains unique advantages.

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.

Crystal structure of a constitutive active mutant of adenosine A2A receptor.

The adenosine A2A receptor (A2AAR) is a prototypical member of the class A subfamily of G-protein-coupled receptors (GPCRs) that is widely distributed in various tissues and organs of the human body, and participates in many important signal-regulation processes. We have previously summarized a common activation pathway of class A GPCRs in which a series of conserved residues/motifs undergo conformational change during extracellular agonist binding and finally induce the coupling of intracellular G protein. Through this mechanism we have successfully predicted several novel constitutive active or inactive mutations for A2AAR. To reveal the molecular mechanism of mutation-induced constitutive activity, we determined the structure of a typical mutant I92N complexed with the agonist UK-432097. The mutated I92N forms a hydrophilic interaction network with nearby residues including Trp6.48 of the CWxP motif, which is absent in wild-type A2AAR. Although the mutant structure is similar overall to the previously determined intermediate-state A2AAR structure (PDB ID 3qak) [Xu, Wu, Katritch, Han, Jacobson, Gao, Cherezov & Stevens (2011). Science, 332, 322-327 ▸], molecular dynamics simulations suggest that the I92N mutant stabilizes the metastable intermediate state through the hydrophilic interaction network and favors the conformational transition of the receptor towards the active state. This research provides a structural template towards the special pharmacological outcome triggered by conformational mutation and sheds light on future structural or pharmaco-logical studies among class A GPCRs.

Determining Sequence-Dependent DNA Oligonucleotide Hybridization and Dehybridization Mechanisms Using Coarse-Grained Molecular Simulation, Markov State Models, and Infrared Spectroscopy.

A robust understanding of the sequence-dependent thermodynamics of DNA hybridization has enabled rapid advances in DNA nanotechnology. A fundamental understanding of the sequence-dependent kinetics and mechanisms of hybridization and dehybridization remains comparatively underdeveloped. In this work, we establish new understanding of the sequence-dependent hybridization/dehybridization kinetics and mechanism within a family of self-complementary pairs of 10-mer DNA oligomers by integrating coarse-grained molecular simulation, machine learning of the slow dynamical modes, data-driven inference of long-time kinetic models, and experimental temperature-jump infrared spectroscopy. For a repetitive ATATATATAT sequence, we resolve a rugged dynamical landscape comprising multiple metastable states, numerous competing hybridization/dehybridization pathways, and a spectrum of dynamical relaxations. Introduction of a G:C pair at the terminus (GATATATATC) or center (ATATGCATAT) of the sequence reduces the ruggedness of the dynamics landscape by eliminating a number of metastable states and reducing the number of competing dynamical pathways. Only by introducing a G:C pair midway between the terminus and the center to maximally disrupt the repetitive nature of the sequence (ATGATATCAT) do we recover a canonical “all-or-nothing” two-state model of hybridization/dehybridization with no intermediate metastable states. Our results establish new understanding of the dynamical richness of sequence-dependent kinetics and mechanisms of DNA hybridization/dehybridization by furnishing quantitative and predictive kinetic models of the dynamical transition network between metastable states, present a molecular basis with which to understand experimental temperature jump data, and furnish foundational design rules by which to rationally engineer the kinetics and pathways of DNA association and dissociation for DNA nanotechnology applications.

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca2+-ATPase

Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca2+, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca2+ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca2+ ions and protons when the Ca2+ ions are released toward the SR lumen.

Alex Proekt, M.D., Ph.D., Recipient of the 2021 James E. Cottrell, M.D., Presidential Scholar Award

Alex Proekt, M.D., Ph.D., Recipient of the 2021 James E. Cottrell, M.D., Presidential Scholar Award