Long-lived Intermediate State Research Articles

Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-μs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

AN UNUSUAL PARAGENESIS OF DIAMOND, GRAPHITE, AND CALCITE: A RAMAN SPECTROSCOPIC STUDY

This work presents a study of an unusual synthetic diamond sample, where calcite is the main phase in addition to diamond. Using Raman spectroscopy it can be shown that in addition to diamond carbon forms four different generations: highly ordered graphite-II, lower ordered graphite-I as spandrel between different mineral grains, weakly-ordered carbonaceous material as graphite-III and the hexagonal diamond lonsdaleite (here graphite-IV) as last formation in calcite. Graphite-III can be found mainly in the calcite body. According to the fine dispersion of this carbonaceous material and the arrangement on grain boundaries, we assume that carbon was dissolved in the calcite melt, and that by the activated state of carbon in the calcite melt the formation of diamond is favored near 1760°C, and 6.8 GPa. Lonsdaleite as minor phase may have been formed as an longlived intermediate state under standard conditions between graphite and diamond. Evidence shows that there are two different lonsdaleite phases (possibly hexagonal and monocline) present. The prevailing diamond is characterized by the first-order Raman line at 1333 cm-1. However, there are also present diamonds with the first-order Raman line down to 1310.6 cm-1, corresponding to 13C = 0.511. Significantly there is the strong decrease of the optical damage threshold with increase of the 13C content.

Fermionic dark matter via UV and IR freeze-in and its possible X-ray signature

Non-observation of any dark matter signature at various direct detection experiments over the last decade keeps indicating that immensely popular WIMP paradigm may not be the actual theory of particle dark matter. Non-thermal dark matter produced through freeze-in is an attractive proposal, naturally explaining null results by virtue of its feeble couplings with the Standard Model (SM) particles. We consider a minimal extension of the SM by two gauge singlet fields namely, a ℤ2-odd fermion χ and a pseudo scalar ̃ϕ, where the former has interactions with the SM particles only at dimension five level and beyond. This introduces natural suppression in the interactions of χ by a heavy new physics scale Λ and forces χ to be a non-thermal dark matter candidate. We have studied the production of χ in detail taking into account both ultra-violate (UV), infra-red (IR) as well as mixed UV-IR freeze-in and found that for 1010 GeV ⩽ Λ⩽ 1015 GeV, χ is dominantly produced via UV and mixed UV-IR freeze-in when reheat temperature TRH≳ 104 GeV and below which the production is dominated by IR and mixed freeze-in. Furthermore, we have considered the cascade annihilation χ χ̄ → ̃ϕ̃ϕ→ 4γ to address the longstanding ∼ 3.5 keV X-ray line observed from various galaxies and galaxy clusters. We have found that the long-lived intermediate state ̃ϕ modifies dark matter density around the galactic centre to an effective density ρeff which strongly depends on the decay length of ̃ϕ. Finally, the allowed parameter space in Λ–g plane (g is the coupling between χχ̄ and ̃ϕ) is obtained by comparing our result with the XMM Newton observed X-ray flux from the centre of Milky Way galaxy in 2σ range.

Dual coherent and incoherent two-photon luminescence in single gold nanorods revealed by polarization and time-resolved nonlinear autocorrelation

It is well known that gold nanorods (AuNRs) readily emit two-photon luminescence (TPL) when excited by a broad bandwidth laser pulse that is tuned to the AuNRs’ localized surface plasmon resonance. The nature of the mechanism (i.e., especially its degree of coherence) is under active debate. In this work, we measured the TPL emission from single nanorods while varying the angle θ between the linearly polarized laser electric field and the nanorod’s orientation. Data were best fit with a linear combination of cos4 θ and cos2 θ functions. While the former function may represent TPL signals arising from both coherent and incoherent processes, the later function is indicative of a purely incoherent process. To further validate this assessment, we measured TPL emission from single nanorods in a time-resolved collinear autocorrelation setup. The autocorrelation signal exhibited a large peak at zero delay, which is characteristic of coherent two-photon absorption and two lower intensity wings extending to a few picoseconds, which demonstrates the existence of a long-lived intermediate state that contributes to a two-step incoherent absorption process. We conclude that TPL in AuNRs can result from a combination of coherent and incoherent absorption processes.

Unexpected Intermediate State Photoinduced in the Metal-Insulator Transition of Submicrometer Phase-Separated Manganites.

At ultrafast timescales, the initial and final states of a first-order metal-insulator transition often coexist forming clusters of the two phases. Here, we report an unexpected third long-lived intermediate state emerging at the photoinduced first-order metal-insulator transition of La_{0.325}Pr_{0.3}Ca_{0.375}MnO_{3}, known to display submicrometer length-scale phase separation. Using magnetic force microscopy and time-dependent magneto-optical Kerr effect, we determined that the third state is a nanoscale mixture of the competing ferromagnetic metallic and charge-ordered insulating phases, with its own physical properties. This discovery bridges the two different families of colossal magnetoresistant manganites known experimentally and shows for the first time that the associated states predicted by theory can coexist in a single sample.

Melting of large Pt@MgO(1 0 0) icosahedra

On the basis of ab initio calculations, we present a new parametrisation of the Vervisch–Mottet–Goniakowski (VMG) potential (Vervisch et al 2002 Phys. Rev. B 24 245411) for modelling the oxide–metal interaction. Applying this model to mimic the finite temperature behaviour of large platinum icosahedra deposited on the pristine MgO(1 0 0), we find the nanoparticle undergoes two solid–solid transitions. At 650 K the ‘squarisation’ of the interface layer, while a full reshaping towards a fcc architecture takes place above 950 K. In between, a quite long-lived intermediate state with a (1 0 0) interface but with an icosahedral cap is observed. Our approach reproduces experimental observations, including wetting behaviour and the lack of surface diffusion.

Conformational Dynamics of Cas9 during DNA Binding

Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA using two conserved nuclease domains, RuvC and HNH, as part of CRISPR-Cas bacterial adaptive immune systems. Together with single-guide RNAs, Cas9 is also widely utilized as a programmable genome editing tool. DNA cleavage activity is controlled directly by the conformational state of the HNH nuclease domain, but Cas9 conformational dynamics during DNA binding remain poorly characterized. Using single-molecule FRET assays, we identified a long-lived intermediate state of S. pyogenes Cas9. Upon DNA binding, the HNH nuclease reversibly transitions between RNA-bound, DNA-bound and docked conformations, before it docks irreversibly into its catalytically active conformation for DNA cleavage. Docking of HNH to its active state requires divalent cation, and HNH remains in the docked state after cleavage of a complementary target. Increasing the number of mutations in the target region distal to the protospacer adjacent motif (PAM) prevents transitions from intermediate to the docked conformation. The results provide a structural explanation for the lack of DNA cleavage activity when Cas9 binds to off-target sites.

Slow Relaxation of Surface Plasmon Excitations in Au55: The Key to Efficient Plasmonic Heating in Au/TiO2.

Gold nanoparticles distinguish themselves from other nanoparticles due to their unique surface plasmon resonance properties that can be exploited for a multiplicity of applications. The promise of plasmonic heating in systems of Au nanoparticles on transition metal oxide supports, for example, Au/TiO2, rests with the ability of the surface plasmon in Au nanoparticles to effectively transfer energy into the transition metal oxide. Here, we report a critical observation regarding Au nanoparticle (Au55) surface plasmon excitations, that is, the relaxation of the surface plasmon excitation is very slow, on the order of several picoseconds. Starting from five plasmon states in Au55 nanoparticles using nonadiabatic molecular dynamics simulations, we find that the relaxation time constant resulting from these simulations is ∼6.8 ps, mainly resulting from a long-lived intermediate state found at around -0.8 eV. This long-lived intermediate state aligns with the conduction band edge of TiO2, thereby facilitating energy transfer injection from the Au55 nanoparticle into the TiO2. The current results rule out the previously reported molecular-like relaxation dynamics for Au55.

Single Spins Charge Ahead

In nanoscale devices like a quantum dot, the presence of even a single excess electron can change the material’s electrical or optical properties. Similarly, the charge on biological macromolecules, such as DNA, affects their interactions with other molecules. Researchers are therefore searching for new ways to sense localized charges on materials in a variety of environments. In Physical Review Letters, Jorg Wrachtrup at the University of Stuttgart, Germany, and his collaborators report a prototype electrometer consisting of an atom-sized defect in diamond, called a nitrogen-vacancy center (NV), that is capable of sensing a single charge on a nearby nitrogenvacancy center. The electrometer could, in principle, work at room temperature and atmospheric pressure and requires no electrical contact with the sample [1]. The technique builds on the group’s earlier work, in which they established that the resonant frequency of a NV’s spin precessing in a magnetic field is influenced by an applied electric field [2]. They now show that this spin resonance signal is sensitive enough to detect the electric field from a single electron 25 nanometers (nm) away. Several methods for sensing individual charges already exist. For example, electrostatic force microscopy [3] and scanning single-electron transistor microscopy [4] have provided astonishing nanoscale views of the distribution of charge states in ceramics and semiconductor devices. A new sensor based on spin resonance is an appealing complement because it requires no electrical connection to the sample, can operate under ambient conditions, and can potentially tolerate environments, such as liquids or harsh chemicals, that are inhospitable to other techniques. In fact, NV center spins have already been pressed into service as nanoscale magnetic-field sensors that can detect a single spin [5]. They can also function as sensitive thermometers [6] that work inside living cells [7]. The NV center is a point defect in diamond that consists of a missing carbon atom (a vacancy) and a nitrogen atom that substitutes for a carbon atom on an adjacent lattice site. At the defect site, the dangling bonds of the surrounding atoms hybridize and form localized electronic energy levels similar to those of a molecule. When an NV center is negatively charged (denoted NV−), it has a total electronic spin of 1, giving it three possible spin projections (ms = 0, +1, −1). An exciting property of NV− is that after being optically excited to a higherenergy electronic state, its relaxation back to the lowest energy state depends on its spin projection. If the spin projection is ms = 0, it almost always relaxes by emitting a photon. Relaxation for the other two spin projections (ms = ±1) could either involve emission of a photon, or it could involve a long-lived intermediate state, with no visible photon emission. As a result, the intensity of light emitted by the NV− depends on its electronic spin projection, which in turn provides an optical method to detect the changes in the average spin projection that occur in a spin resonance experiment. This property, along with the atom-scale size of the defects and their naturally narrow spin resonance linewidths, makes NV centers attractive as optically addressed nanoscale sensors that use spin resonance to monitor the local environment. In 2011, Wrachtrup and his group showed that a NV center’s spin resonance depends on local electric field [2]. In simple terms, this dependence exists because a nearby electric field changes the symmetry of the nitrogenvacancy center. When no electric field is present, the four dangling bonds around the vacancy have roughly the shape of a camera tripod (three bonds form the legs and one bond forms the pole supporting the camera). But in an electric field, the tripod becomes slightly distorted. As a result, the nitrogen-vacancy center’s electron density and spin energies shift, which the researchers can detect as a shift in the spin resonance frequency. The researchers have now shown that the vacancy is

Pyrite multiple-sulfur isotope evidence for rapid expansion and contraction of the early Paleoproterozoic seawater sulfate reservoir

Earthʼs oxygenation is often described in terms of two unidirectional steps at the beginning and end of the Proterozoic Eon, separated by a long-lived intermediate redox state. Recent work defines a more complicated path to oxygenation, exemplified by an apparent drop in oxidation state following the early Paleoproterozoic Lomagundi carbon isotope excursion. The timing of this proposed drop in oxidation state is not well constrained, and it is not clear how it relates to redox conditions during the remainder of the Proterozoic. Here we present a study of pyrite multiple-sulfur isotopes, supported by Fe speciation and organic carbon isotopes, from early Paleoproterozoic black shales. We find evidence for the rapid expansion of the seawater sulfate reservoir during the Great Oxidation Event at ca. 2.3 Ga followed by a subsequent contraction in the size of the seawater sulfate reservoir at ca. 2.05 Ga. This scenario is consistent with the emerging view of a rise and fall in surface oxidation state during the early Paleoproterozoic. Comparison of our new data to other records of the seawater sulfate reservoir suggests that the elevated sulfate concentrations that characterize the early Paleoproterozoic did not return until the late Neoproterozoic.

Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.

The fractal globule as a model of chromatin architecture in the cell.

The fractal globule is a compact polymer state that emerges during polymer condensation as a result of topological constraints which prevent one region of the chain from passing across another one. This long-lived intermediate state was introduced in 1988 (Grosberg et al. 1988) and has not been observed in experiments or simulations until recently (Lieberman-Aiden et al. 2009). Recent characterization of human chromatin using a novel chromosome conformational capture technique brought the fractal globule into the spotlight as a structural model of human chromosome on the scale of up to 10 Mb (Lieberman-Aiden et al. 2009). Here, we present the concept of the fractal globule, comparing it to other states of a polymer and focusing on its properties relevant for the biophysics of chromatin. We then discuss properties of the fractal globule that make it an attractive model for chromatin organization inside a cell. Next, we connect the fractal globule to recent studies that emphasize topological constraints as a primary factor driving formation of chromosomal territories. We discuss how theoretical predictions, made on the basis of the fractal globule model, can be tested experimentally. Finally, we discuss whether fractal globule architecture can be relevant for chromatin packing in other organisms such as yeast and bacteria.

Photodissociation of pyrrole–ammonia clusters by velocity map imaging: mechanism for the H-atom transfer reaction

The photodissociation dynamics of pyrrole-ammonia clusters (PyH·(NH(3))(n), n = 2-6) has been studied using a combination of velocity map imaging and non-resonant detection of the NH(4)(NH(3))(n-1) products. The excited state hydrogen-atom transfer mechanism (ESHT) is evidenced through delayed ionization and presents a threshold around 236.6 nm, in agreement with previous reports. A high resolution determination of the kinetic energy distributions (KEDs) of the products reveals slow (∼0.15 eV) and structured distributions for all the ammonia cluster masses studied. The low values of the measured kinetic energy rule out the existence of a long-lived intermediate state, as it has been proposed previously. Instead, a direct N-H bond rupture, in the fashion of the photodissociation of bare pyrrole, is proposed. This assumption is supported by a careful analysis of the structure of the measured KEDs in terms of a discrete vibrational activity of the pyrrolyl co-fragment.

Phenomenology of dark matter annihilation into a long-lived intermediate state

We propose a scenario where Dark Matter (DM) annihilates into an intermediate state which travels a distance λ ≡ v/Γ on the order of galactic scales and then decays to Standard Model (SM) particles. The long lifetime disperses the production zone of the SM particles away from the galactic center and hence, relaxes constraints from gamma ray observations on canonical annihilation scenarios. We utilize this set up to explain the electron and positron excesses observed recently by PAMELA, ATIC and FERMI. While an explanation in terms of usual DM annihilations seems to conflict with gamma ray observations, we show that within the proposed scenario, the PAMELA/ATIC/FERMI results are consistent with the gamma ray data. The distinction from decay scenarios is discsussed and we comment on the prospects for DM production at LHC. The typical decay length λ ≳ 10 kpc of the intermediate state can have its origin from a dimension six operator suppressed by a scale Λ ∼ 1013 GeV, which is roughly the seesaw scale for neutrino masses.

Force-induced unfolding of human telomeric G-quadruplex: A steered molecular dynamics simulation study

We study the unfolding of a parallel G-quadruplex from human telomeric DNA by mechanical stretching using steered molecular dynamics (MD) simulation. We find that the force curves and unfolding processes strongly depend on the pulling sites. With pulling sites located on the sugar-phosphate backbone, the force-extension curve shows a single peak and the unfolding proceeds sequentially. Pulling sites located on the terminal nucleobases lead to a force-extension curve with two peaks and the unfolding is more cooperative. Simulations of the refolding of partially unfolded quadruplexes show very different behavior for the two different pulling modalities. In particular, starting from an unfolded state prepared by nucleobase pulling leads to a long-lived intermediate state whose existence is also corroborated by the free energy profile computed with the Jarzynski equation. Based on this observation, we propose a novel folding pathway for parallel G-quadruplexes with the human telomere sequence.

Ultrafast dynamics and temperature effects on the quantum efficiency of the ring-opening reaction of a photochromic indolylfulgide

The ultrafast ring-opening reaction of the molecular switch 1,2-Dimethyl-3-indolylfulgide dissolved in acetonitrile is investigated by temperature dependent quantum efficiency measurements and time-resolved transient absorption spectroscopy in the ultraviolet and visible spectral range. The photoreaction is found to be thermally activated with an activation energy of about 1640 cm − 1 . The transient absorption signal is bi-exponential with the time constants τ 1 = 0.7 ps and τ 2 = 12 ps. The fast time constant is due to solvation dynamics, while the main component τ 2 is attributed to the excited state lifetime and product formation. A long-lived intermediate state in the photoreaction can be excluded.

Decay ofC10excited states above the2p+2αthreshold and the contribution from “democratic” two-proton emission

The decay of $^{10}\mathrm{C}$ excited states to the $2p+2\ensuremath{\alpha}$ exit channel has been studied using an $E/A=10.7$ MeV $^{10}\mathrm{C}$ beam inelastically scattered from a $^{9}\mathrm{Be}$ target. Levels associated with two-proton decay to the ground state of $^{8}\mathrm{Be}$ have been observed. These include states at 5.18 and 6.54 MeV which decay by sequential two-proton emission through the long-lived ground state of $^{9}\mathrm{B}$. In addition, states at 5.3 and 6.57 MeV were found in which there is no long-lived intermediate state between the two proton emissions. For the 6.57 MeV state, the two protons are preferably emitted on the same side of the decaying $^{10}\mathrm{C}$ fragment.

Kinetics of disorder-to-fcc phase transition via an intermediate bcc state

Time-resolved small-angle x-ray scattering measurements reveal that a long-lived intermediate bcc state forms when a poly(styrene-b-isoprene) diblock copolymer solution in an isoprene selective solvent is rapidly cooled from the disordered micellar fluid at high temperature to an equilibrium fcc state. The kinetics of the epitaxial growth of the [111] fcc peak from the [110] bcc peak was obtained by fitting the scattering data to a simple model of the transformation. The growth of the [111] fcc peak agrees with the Avrami model of nucleation and growth kinetics with an exponent n=1.4, as does the initial decay of the [110] bcc peak, with an exponent n=1.3. The data were also found to be in good agreement with the Cahn model of grain boundary nucleation and growth.

Primary Photoexcitations and the Origin of the Photocurrent in Rubrene Single Crystals

By simultaneously measuring the excitation spectra of transient luminescence and transient photoconductivity after picosecond pulsed excitation in rubrene single crystals, we show that free excitons are photoexcited starting at photon energies above 2.0 eV. We observe a competition between photoexcitation of free excitons and photoexcitation into vibronic states that subsequently decays into free carriers, while molecular excitons are instead formed predominantly through the free exciton. At photon energies below 2.25 eV, free charge carriers are created only through a long-lived intermediate state with a lifetime of up to 0.1 ms and no free carriers appear during the exciton lifetime.

Multiple folding mechanisms of protein ubiquitin

Based on the C(alpha) Go-type model, the folding kinetics and mechanisms of protein ubiquitin with mixed alpha/beta topology are studied by molecular dynamics simulations. The relaxation kinetics shows that there are three phases, namely the major phase, the intermediate phase and the slowest minor phase. The existence of these three phases are relevant to the phenomenon found in experiments. According to our simulations, the folding at high temperatures around the folding transition temperature T(f) is of a two-state process, and the folding nucleus is consisted of contacts between the front end of alpha-helix and the turn(4). The folding at low temperature (approximately T = 0.8) is also studied, where an A-state like structure is found lying on the major folding pathway. The appearance of this structure is related to the stability of the first part (residue 1-51) of protein ubiquitin. As the temperature decreases, the formation of secondary structures, tertiary structures and collapse of the protein are found to be decoupled gradually and the folding mechanism changes from the nucleation-condensation to the diffusion-collision. This feature indicates a unifying common folding mechanism for proteins. The intermediate phase is also studied and is found to represent a folding process via a long-lived intermediate state which is stabilized by strong interactions between the beta(1) and the beta(5) strand. These strong interactions are important for the function of protein ubiquitin as a molecular chaperone. Thus the intermediate phase is assumed as a byproduct of the requirement of protein function. In addition, the validity of the current Go-model is also investigated, and a lower limited temperature for protein ubiquitin T(limit) = 0.8 is proposed. At temperatures higher than this value, the kinetic traps due to glass dynamics cannot be significantly populated and the intermediate states can be reliably identified although there is slight chevron rollover in the folding rates. At temperature lower than T(limit), however, the traps due to glass dynamics become dominant and may be mistaken for real intermediate states. This limitation of valid temperature range prevents us to reveal the burst phase intermediate in the major folding phase since it might only be stabilized at temperatures lower than T(limit), according to experiments. Our works show that caution must be taken when studying low-temperature intermediate states by using the C(alpha) Go-models.