Submicrosecond Time Scale Research Articles

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation.

For a long time, the effect of a crowded cellular environment on protein dynamics has been largely ignored. Recent experiments indicate that proteins diffuse more slowly in a living cell than in a diluted solution, and further studies suggest that the diffusion depends on the local surroundings. Here, detailed insight into how diffusion depends on protein-protein contacts is presented based on extensive all-atom molecular dynamics simulations of concentrated villin headpiece solutions. After force field adjustments in the form of increased protein-water interactions to reproduce experimental data, translational and rotational diffusion was analyzed in detail. Although internal protein dynamics remained largely unaltered, rotational diffusion was found to slow down more significantly than translational diffusion as the protein concentration increased. The decrease in diffusion is interpreted in terms of a transient formation of protein clusters. These clusters persist on sub-microsecond time scales and follow distributions that increasingly shift toward larger cluster size with increasing protein concentrations. Weighting diffusion coefficients estimated for different clusters extracted from the simulations with the distribution of clusters largely reproduces the overall observed diffusion rates, suggesting that transient cluster formation is a primary cause for a slow-down in diffusion upon crowding with other proteins.

Pink-beam serial crystallography

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.

Application of Pulse Radiolysis to Mechanistic Investigations of Catalysis Relevant to Artificial Photosynthesis.

Taking inspiration from natural photosystems, the goal of artificial photosynthesis is to harness solar energy to convert abundant materials, such as CO2 and H2 O, into solar fuels. Catalysts are required to ensure that the necessary redox half-reactions proceed in the most energy-efficient manner. It is therefore critical to gain a detailed mechanistic understanding of these catalytic reactions to develop new and improved catalysts. Many of the key catalytic intermediates are short-lived transient species, requiring time-resolved spectroscopic techniques for their observation. The two main methods for rapidly generating such species on the sub-microsecond timescale are laser flash photolysis and pulse radiolysis. These methods complement one another, and both provide important spectroscopic and kinetic information. However, pulse radiolysis proves to be superior in systems with significant spectroscopic overlap between the photosensitizer and other species present during the reaction. Herein, the pulse radiolysis technique and how it has been applied to mechanistic investigations of halfreactions relevant to artificial photosynthesis are reviewed.

Sub-microsecond temporal evolution of edge density during edge localized modes in KSTAR tokamak plasmas inferred from ion cyclotron emission

During edge localised mode (ELM) crashes in KSTAR deuterium plasmas, bursts of spectrally structured ion cyclotron emission (ICE) are detected. Usually the ICE spectrum chirps downwards during an ELM crash, on sub-microsecond timescales. For KSTAR ICE where the separation of spectral peak frequencies is close to the proton cyclotron frequency at the outer plasma edge, we show that the driving population of energetic ions is likely to be a subset of the 3 MeV fusion protons, born centrally on deeply passing orbits which drift from the core to the edge plasma. We report first principles modelling of this scenario using a particle-in-cell code, which evolves the full orbit dynamics of large numbers of energetic protons, thermal deuterons, and electrons self-consistently with the electric and magnetic fields. The Fourier transform of the excited fields in the nonlinear saturated regime of the simulations is the theoretical counterpart to the measured ICE spectra. Multiple simulation runs for different, adjacent, values of the plasma density under KSTAR edge conditions enable us to infer the theoretical dependence of ICE spectral structure on the local electron number density. By matching this density dependence to the observed time-dependence of chirping ICE spectra in KSTAR, we obtain sub-microsecond time resolution of the evolving local electron number density during the ELM crash.

Fast Reconfigurable SOA-Based Wavelength Conversion of Advanced Modulation Format Data

We theoretically analyze the phase noise transfer issue between the pump and the wavelength-converted idler for a nondegenerate four-wave mixing (FWM) scheme, as well as study the vector theory in nonlinear semiconductor optical amplifiers (SOAs), in order to design a polarization-insensitive wavelength conversion system employing dual co-polarized pumps. A tunable sampled grating distributed Bragg reflector (SG-DBR) pump laser has been utilized to enable fast wavelength conversion in the sub-microsecond timescale. By using the detailed characterization of the SGDBR laser, we discuss the phase noise performance of the SGDBR laser. Finally, we present a reconfigurable SOA-based all-optical wavelength converter using the fast switching SGDBR tunable laser as one of the pump sources and experimentally study the wavelength conversion of the single polarization quadrature phase shift keying (QPSK) and polarization multiplexed (Pol-Mux) QPSK signals at 12.5-Gbaud. A wide tuning range (>10 nm) and less than 50 ns and 160 ns reconfiguration time have been achieved for the wavelength conversion system for QPSK and PM-QPSK signals, respectively. The performance under the switching environment after the required reconfiguration time is the same as the static case when the wavelengths are fixed.

A platform for exploding wires in different media

A platform SWE-2 used for single wire explosion experiments has been designed, established, and commissioned. This paper describes the design and initial experiments of SWE-2. In summary, two pulsed current sources based on pulse capacitors and spark gaps are adopted to drive sub-microsecond and microsecond time scale wire explosions in a gaseous/liquid medium, respectively. In the initial experiments, a single copper wire was exploded in air, helium, and argon with a 0.1-0.3 MPa ambient pressure as well as tap water with a 283-323 K temperature, 184-11 000 μS/cm conductivity, or 0.1-0.9 MPa hydrostatic pressure. In addition, the diagnostic system is introduced in detail. Energy deposition, optical emission, and shock wave characteristics are briefly discussed based on experimental results. The platform was demonstrated to operate successfully with a single wire load. These results provide the potential for further applications of this platform, such as plasma-matter interactions, shock wave effects, and reservoir simulations.

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliGC). We have identified point mutations in FliGC from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliGC from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliGC are observed in the Cα1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliGC at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix α1 in the Cα1-6 domain of FliGC contribute to ensure the precise regulation of the motor switching.

Photoisomerization induced scission of rod-like micelles unravelled with multiscale modeling

HypothesisIn photorheological fluids, subtle molecular changes caused by light lead to abrupt macroscopic alterations. Upon UV irradiation of an aqueous cetyltrimethylammonium bromide (CTAB) and trans-ortho-methoxycinnamic acid (trans-OMCA) solution, for instance, the viscosity drops over orders of magnitude. Multiscale modeling allows to elucidate the mechanisms behind these photorheological effects. ExperimentsWe use time-dependent DFT calculations to study the photoisomerization, and a combination of atomistic molecular dynamics (MD) and DFT to probe the influence of both OMCA isomers on the micellar solutions. FindingsThe time-dependent DFT calculations show that the isomerization pathway occurs in the first triplet excited state with a minimum energy conformation closest to the after photoisomerization predominant cis configuration. In the MD simulations, with sub-microsecond timescales much shorter than the experimental morphological transition, already a clear difference is observed in the packing of the two OMCA isomers: contrary to trans-OMCA, cis-OMCA exposes notable part of its hydrophobic aromatic rings at the micelle surface. This can explain why trans-OMCA adopts rod-like micellar packing (high viscosity) while cis-OMCA spherical micellar packing (low viscosity). Moreover, lowering of the OMCA co-solute concentration allowed us to perform full simulation of the breakup process of the rod-like micelles which are stable prior to isomerization.

Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein.

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. To complement the information from FRET, we combine it with photoinduced electron transfer (PET) quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond time scale can be obtained reliably from a single set of measurements by the analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A more detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic molecular dynamics simulation of integrase. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of the dynamics and detailed mechanisms in unfolded and intrinsically disordered proteins.

A RISC-V Processor SoC With Integrated Power Management at Submicrosecond Timescales in 28 nm FD-SOI

This paper presents a RISC-V system-on-chip (SoC) with integrated voltage regulation, adaptive clocking, and power management implemented in a 28 nm fully depleted silicon-on-insulator process. A fully integrated simultaneous-switching switched-capacitor DC–DC converter supplies an application core using a clock from a free-running adaptive clock generator, achieving high system conversion efficiency (82%–89%) and energy efficiency (41.8 double-precision GFLOPS/W) while delivering up to 231 mW of power. A second core serves as an integrated power-management unit that can measure system state and actuate changes to core voltage and frequency, allowing the implementation of a wide variety of power-management algorithms that can respond at submicrosecond timescales while adding just 2.0% area overhead. A voltage dithering program allows operation across a wide continuous voltage range (0.45 V–1 V), while an adaptive voltage-scaling algorithm reduces the energy consumption of a synthetic benchmark by 39.8% with negligible performance penalty, demonstrating practical microsecond-scale power management for mobile SoCs.

Efficient Detection of 3 THz Radiation from Quantum Cascade Laser Using Silicon CMOS Detectors

In this paper, we report on efficient detection of the radiation emitted by a THz quantum cascade laser (QCL) using an antenna-coupled field effect transistor (TeraFET). In the limiting case when all radiated power would be collected, the investigated TeraFET can show up to 230 V/W responsivity with the noise equivalent power being as low as 85 pW/ $\sqrt {\text {Hz}}$ at 3.1 THz, which is several times lower than that of the typical Golay cell. A combination of the QCL and a set of off-axis parabolic mirrors with 3-inch and 2-inch focal lengths was used to measure the signal-to-noise ratio (SNR) of the TeraFET. The practically achieved SNR was five times lower than that of the Golay cell and two orders of magnitude lower than a bolometer’s. However, TeraFETs are much faster and do not need a signal modulation, thus can be used both in a continuous mode for power monitoring or for investigation of transient processes on a sub-microsecond time scale.

Sub-microsecond x-ray imaging using hole-collecting Schottky type CdTe with charge-integrating pixel array detectors

CdTe is increasingly being used as the x-ray sensing material in imaging pixel array detectors for x-rays, generally above 20 keV, where silicon sensors become unacceptably transparent. Unfortunately CdTe suffers from polarization, which can alter the response of the material over time and with accumulated dose. Most prior studies used long integration times or CdTe that was not of the hole-collecting Schottky type. We investigated the temporal response of hole-collecting Schottky type CdTe sensors on timescales ranging from tens of nanoseconds to several seconds. We found that the material shows signal persistence on the timescale of hundreds of milliseconds attributed to the detrapping of a shallow trap, and additional persistence on sub-microsecond timescales after polarization. The results show that this type of CdTe can be used for time resolved studies down to approximately 100 ns. However quantitative interpretation of the signal requires careful attention to bias voltages, polarization and exposure history.

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the pKa value in delipidated HSA is 8.1±0.2 at 37°C and 0.1M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H2O2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous SN2 transition state (TS) with calculated Δ‡G and Δ‡H barriers at 298K of respectively 59 and 54kJmol−1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA–SO− and water as products. The structured reaction site cages H2O2, which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H2O2. The lack of reaction-site features required for differential stabilization of the TS (positive charges, H2O2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA–SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA–SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H2O2.

Energy and charge transfer cascade in methylammonium lead bromide perovskite nanoparticle aggregates.

Highly photoluminescent hybrid lead halide perovskite nanoparticles have recently attracted wide interest in the context of high-stake applications, such as light emitting diodes (LEDs), light emitting transistors and lasers. In addition, they constitute ideal model systems to explore energy and charge transport phenomena occurring at the boundaries of nanocrystalline grains forming thin films in high-efficiency perovskite solar cells (PSCs). Here we report a complete photophysical study of CH3NH3PbBr3 perovskite nanoparticles suspended in chlorobenzene and highlight some important interaction properties. Colloidal suspensions under study were constituted of dispersed aggregates of quasi-2D platelets of a range of thicknesses, decorated with 3D-like spherical nanoparticles. These types of nanostructures possess different optical properties that afford a handle for probing them individually. The photophysics of the colloidal particles was studied by femtosecond pump-probe spectroscopy and time-correlated single-photon counting. We show here that a cascade of energy and exciton-mediated charge transfer occurs between nanostructures: upon photoexcitation, localized excitons within one nanostructure can either recombine on a ps timescale, yielding a short-lived emission, or form charge-transfer states (CTSs) across adjacent domains, resulting in longer-lived photoluminescence in the millisecond timescale. Furthermore, CTSs exhibit a clear signature in the form of a strong photoinduced electroabsorption evidenced in femtosecond transient absorption measurements. Charge transfer dynamics at the surface of the nanoparticles have been studied with various quenchers in solution. Efficient hole transfer to N,N,N',N'-tetrakis(4-methoxyphenyl)benzidine (MeO-TPD) and 1,4-bis(diphenyl-amino)benzene (BDB) donors was attested by the quenching of the nanoparticles emission. The charge transfer rate was limited by the organic layer used to stabilize the nanoparticles, which acted as a wide spacer between reactants. The forward charge transfer was found to take place in the sub-microsecond time-scale in competition with slow carrier recombination, while back transfer was shown to occur with a time-constant τ = 25 ms.

Finite Temperature String Method with Umbrella Sampling: Application on a Side Chain Flipping in Mhp1 Transporter.

Protein conformational change is of central importance in molecular biology. Here we demonstrate a computational approach to characterize the transition between two metastable conformations in all-atom simulations. Our approach is based on the finite temperature string method, and the implementation is essentially a generalization of umbrella sampling simulations with Hamiltonian replica exchange. We represent the transition pathway by a curve in the conformational space, with the curve parameter taken as the reaction coordinate. Our approach can efficiently refine a transition pathway and compute a one-dimensional free energy as a function of the reaction coordinate. A diffusion model can then be used to calculate the forward and backward transition rates, the major kinetic quantities for the transition. We applied the approach on a local transition in the ligand-free Mhp1 transporter, between its outward-facing conformation and an intermediate conformation with the side chain of Phe305 flipped to the outside of the protein. Our simulations predict that the flipped-out position of this side chain has a free energy 6.5 kcal/mol higher than the original position in the crystal structure, and that the forward and backward transition rates are in the millisecond and submicrosecond time scales, respectively.

Electric-field-stimulated protein mechanics.

The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

Molecular Mechanism Behind the Fast Folding/Unfolding Transitions of Villin Headpiece Subdomain: Hierarchy and Heterogeneity.

Proteins involve motions over a wide range of spatial and temporal scales. While the large conformational changes, such as folding and functioning, are slow and appear to occur in a highly cooperative manner, how the hierarchical dynamics over different time scales play a role during these slow transitions has been of great interest over the decades. Here we study the folding mechanism of the villin headpiece subdomain (HP35) to understand the molecular mechanism behind this prototypical fast-folding protein. The ∼400 μs molecular dynamics (MD) trajectories obtained by Piana et al. [ Piana , S. ; Lindorff-Larsen , K. ; Shaw , D. E. Proc. Natl. Acad. Sci. U.S.A. 2012 , 109 , 17845 ] are analyzed in detail. By extracting the slowest mode from the trajectories, which is responsible for the folding/unfolding transitions, and by analyzing the transition events along this mode, we find that the transitions occur in a heterogeneous manner. Detailed analysis of the individual transition events shows that the folding/unfolding transitions occur via two qualitatively different pathways, i.e., the unfolding triggered from the C-terminal (α3 helix) and from the N-terminal (α1-α2 loop). Non-native contacts are also found to contribute in slowing down the transitions. The folding of HP35 thus proceeds in a segmental manner rather than cooperatively at the submicrosecond time scale. The Lys→Nle mutation is found to speed up the transitions by rigidifying the α3 helix, i.e., suppressing one transition pathway. The analysis of the microsecond dynamics in the single-molecule Förster resonance energy transfer efficiency trajectories, which are calculated from the MD data, reveals that the folding/unfolding transitions in the NleNle mutant can be fitted with a two-state model, whereas those in WT appear to be more complex and involves multiple time scales. This is due to the coupling between the folding/unfolding transitions and conformational transitions within the unfolded and intermediate states. The present study demonstrates that a protein as small as HP35 already involves heterogeneous characters during folding/unfolding transitions when the hierarchical dynamics at the molecular level is considered, thus heterogeneity can be a general characteristic in protein folding.

Nonclassical Crystal Growth as Explanation for the Riddle of Polarity in Centrosymmetric Glycine Crystals

The riddle of anomalous polar behavior of the centrosymmetric crystal of α-glycine is resolved by the discovery of a polar, several hundred nanometer thick hydrated layer, created at the {010} faces during crystal growth. This layer was detected by two independent pyroelectric analytical methods: (i) periodic temperature change technique (Chynoweth) at ambient conditions and (ii) contactless X-ray photoelectron spectroscopy under ultrahigh vacuum. The total polarization of the surface layer is extremely large, yielding ≈1 μC·cm-2, and is preserved in ultrahigh vacuum, but disappears upon heating to 100 °C. Molecular dynamics simulations corroborate the formation of polar hydrated layers at the sub-microsecond time scale, however with a thickness of only several nanometers, not several hundred. This inconsistency might be reconciled by invoking a three-step nonclassical crystal growth mechanism comprising (i) docking of clusters from the supersaturated solution onto the evolving crystal, (ii) surface recognition and polar induction, and (iii) annealing and dehydration, followed by site-selective recrystallization.

Conformational Thermodynamics of DNA Strands in Hydrophilic Nanopores

Enhanced sampling techniques spanning a submicrosecond time scale reveal that a double-stranded DNA dodecamer can be spontaneously encapsulated into (51, 0) and (40, 0) single-walled carbon nanotubes under the influence of an electric field, leading to hybrids with a 40 kJ/mol enhanced free-energy. The confinement mechanism allows the nucleic acid to retain its mobility, diffusing anisotropically along the endohedral volume, visiting regions of space determined by entropic factors (diameter, free volume) and linked by a thermodynamical highway. In spite of the energetic similarities between both topologies (4.1 × 103 kJ/mol), the biomolecule favors positioning either parallel to the nanopore central axis (40, 0) or in close contact with the solid walls (51, 0), encasing a hollow cylindrical domain of diameter 1–1.5 nm in the latter. Precise physiological conditions allow the extrapolation of results to in vivo systems and constitute a novel and thorough contribution to nanotube technology in the areas of ...

Intramolecular Electron Transfer from Tryptophan to Guanosyl Radicals in a Linked System as a Model of DNA Repair

Abstract As a model of chemical DNA repair, intramolecular electron transfer from tryptophan to the radical of the purine base guanosine combined into a conjugate by a flexible linker was studied by time-resolved chemically induced dynamic nuclear polarization (CIDNP). The guanosyl radicals were photochemically generated in the quenching reaction of the triplet excited dye 2,2′-dipyridyl. The CIDNP kinetics was obtained by detection of NMR spectra containing anomalously enhanced signals of diamagnetic products that are formed during a variable period after excitation by a laser pulse. The kinetic data obtained for the protons located on the guanosyl and tryptophanyl moieties of the conjugate were compared to those obtained in photoreactions of the molecules containing the same linker, but with only one of the two reactive moieties of the conjugate – tryptophanyl or guanosyl. Strong differences between the CIDNP kinetics of different conjugates were revealed and explained by a rapid intramolecular electron transfer from tryptophan to the guanosyl radical in the conjugate. Model simulations of the CIDNP kinetics allowed for determination of the rate constant of intramolecular electron transfer at (1.0±0.5)×106 s–1 indicating a high potential of chemical repair of the guanosyl radical by means of electron transfer from the tryptophanyl moiety in a surrounding protein pool that can provide rather efficient protection of oxidized DNA bases from pathological damage on a submicrosecond time scale.