Time-resolved Circular Dichroism Research Articles

Ultrafast demagnetization in buried Co80Dy20 as fingerprint of hot-electron transport

Abstract The generation of ultrashort hot-electron pulses in metallic heterostructures offers attractive perspectives for the ultrafast spin manipulation on the picosecond time scale. In such approach, the hot-electron pulses are produced by exciting a non-magnetic capping layer with femtosecond infrared laser pulses. These hot-electron pulses propagate towards a buried magnetic layer to trigger ultrafast demagnetization. Lately, it was shown that the demagnetization onset and characteristic demagnetization times are both affected by the transport regime (ballistic or diffusive) of the photo-excited hot-electrons. In this work, we show that the hot-electron pulses produced by photo-exciting a Al(3)/Ta(3)/Cu(60) capping layer undergo a temporal stretching and delays when they go across a [Co(0.1)/Ni(0.6)]x5 multilayer. These information were extracted from a study of hot-electron induced demagnetization in CoDy alloys by means of Time-Resolved X-Ray Magnetic Circular Dichroism.

Spectroscopic Insight on Ethanol-Induced Aggregation of Papain.

In this contribution, the structural and dynamic changes occurring to papain in ethanol-water binary solvent mixtures have been investigated and compared with its denatured state. Steady-state fluorescence, solvation dynamics, time-resolved rotational anisotropy, circular dichroism (CD), and single molecular-level fluorescence correlation spectroscopic (FCS) studies were performed for this purpose. In ethanol-water mixtures with XEtOH = 0.6, N-(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide (DACIA)-tagged papain was found to undergo a blue shift of 12 nm, while in the presence of 5 M GnHCl, a red shift of 5 nm was observed. Solvation dynamics of the system was also found to be different in the presence of these external agents. In ethanol-water mixtures, the average solvation time was found to increase almost 2-fold as compared to that in water, while in the presence of GnHCl, only a marginal increase could be observed. These changes of DACIA-tagged papain in ethanol-water mixtures are attributed to the aggregation of the protein in the presence of ethanol. The residual anisotropy was found to increase 14-fold, and the rotational time component corresponding to the rotation of the probe molecule was found to increase by 4-fold in the ethanol-water mixture which also gives a notion of the papain aggregation. Atomic force microscopy (AFM) confirms this aggregate formation, which is also quantified by the FCS study. The hydrodynamic radius of the protein aggregates in ethanol-water mixtures was calculated to be ∼155 Å as compared to the corresponding value of 18.4 Å in the case of native monomer papain. Also, it confirmed that the aggregate formation takes place even in the nanomolar concentration of papain. Analysis of circular dichroism spectra of papain showed that an increase in the β-sheet content of papain at the expense of α-helix and the random coil with an increase of the ethanol mole fraction may be responsible for this aggregation process.

Exploring the Structural Properties of Synaptotagmin's Intrinsically Disordered Region

We used time-resolved FRET, circular dichroism, and all-atom simulation to investigate the structural impact of phosphorylation and dielectric constant on synaptotagmin 1's intrinsically disordered region (IDR). We found that the full-length IDR sequence, a ∼60 residue strong polyampholyte that when studied in the form of a peptide, undergoes structural collapse consistent with its κ-predicted behavior. Furthermore, we found that the exocytosis-modulating phosphorylation of Thr112, a residue located in the IDR's more sequence diverse central core region, disrupts a local disorder-to-order transition that occurs when solution dielectric constant is lowered and helical structure is stabilized by addition of trifluoroethanol. Implicit solvent simulations testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric, where several lysine-aspartic acid salt bridges stabilize transient secondary structure. Phosphorylation, however, results in formation of salt bridges unsuitable for helix formation. These results suggest a model where compaction of the IDR sequence and phosphorylation may regulate structural transitions that in turn modulate neuronal exocytosis.

Ultrafast broadband circular dichroism in the deep ultraviolet

The measurement of chirality and its temporal evolution are crucial for the understanding of a large range of biological functions and chemical reactions. Steady-state circular dichroism (CD) is a standard analytical tool for measuring chirality in chemistry and biology. Nevertheless, its push into the ultrafast time domain and in the deep-ultraviolet has remained a challenge, with only some isolated reports of subnanosecond CD. Here, we present a broadband time-resolved CD spectrometer in the deep ultraviolet (UV) spectral range with femtosecond time resolution. The setup employs a photoelastic modulator to achieve shot-to-shot polarization switching of a 20 kHz pulse train of broadband femtosecond deep-UV pulses (250–370 nm). The resulting sequence of alternating left- and right-circularly polarized probe pulses is employed in a pump-probe scheme with shot-to-shot dispersive detection and thus allows for the acquisition of broadband CD spectra of ground- and excited-state species. Through polarization scrambling of the probe pulses prior to detection, artifact-free static and transient CD spectra of enantiopure [Ru(bpy)3]2+ are successfully recorded with a sensitivity of <2×10−5 OD (≈0.7 mdeg). Due to its broadband deep-UV detection with unprecedented sensitivity, the measurement of ultrafast chirality changes in biological systems with amino-acid residues and peptides and of DNA oligomers is now feasible.

Proposal of X-ray absorption spectroscopy and magnetic circular dichroism using broadband free-electron lasers.

X-ray free-electron lasers (XFELs) have been widely used for applications such as X-ray crystallography and magnetic spin probes because of their unprecedented performance. Recently, time-resolved X-ray magnetic circular dichroism (XMCD) with ultrafast XFEL pulses have made it possible to achieve an instantaneous view of atomic de-excitation. However, owing to the narrow bandwidth and coherence of XFELs, X-ray absorption spectroscopy (XAS) and XMCD are time- and effort-consuming for both machine scientists and users of XFELs. In this work, an efficient scheme using a broadband XFEL pulse and single-shot X-ray spectrometer is proposed, in which the XAS and XMCD measurements can be accomplished with the same machine conditions. An evolutionary multi-objective optimization algorithm is used to maximize the XFEL bandwidth offered by the Shanghai Soft X-ray FEL user facility without additional hardware. A numerical example using MnO is demonstrated, showing that, by using approximately 1000 consecutive XFEL shots with a central photon energy of 650 eV and full bandwidth of 4.4%, precise spectral measurements for XAS and XMCD can be achieved. Additional considerations related to single-shot XAS and XMCD are discussed.

Synthesis, characterization and unravelling the molecular interaction of new bioactive 4-hydroxycoumarin derivative with biopolymer: Insights from spectroscopic and theoretical aspect

In the progress of small molecule as drug candidates, 4-hydroxycoumarin based compounds bearing a crucial place as potent antibiotic agents with appreciable safety in drug invention. Being synthetically and easily obtainable, 4-hydroxycoumarin related compounds with planar structure have been promoted predominantly as DNA targeting agent. Nevertheless, here we elucidate the synthesis, characterization and theoretical study of bio-active small molecule 4-hydroxy-3,4′-bichromenyl-2,2′-dione (4HBD). Then we have illuminated the binding interactions of 4HBD with calf thymus DNA (ctDNA), which is particularly designed for biological application. Extensive investigations of the binding of 4HBD with ctDNA are provided by utilizing multi-spectroscopic and molecular docking approaches, including UV–vis absorbance, steady-state, time-resolved fluorescence spectroscopy and circular dichroism study. The calculated binding and quenching constant value from quantitative data analysis of absorption and emission spectroscopy shows that 4HBD binds to the ctDNA groove. Further confirmation of the same is found by comparative displacement and iodide quenching studies. Negative enthalpy, negative free energy and positive entropy change imply a hydrophobic force monitors the association of 4HBD with the biomacromolecule. Interestingly the small molecule (4HBD) shows potential anti-bacterial activity against the model pathogenic gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria. The noncytotoxic nature of the 4HBD is demonstrated in vitro with the help of MTT assay by normal kidney epithelial (NKE), breast cancer cells (MCF-7) and human prostate cancer cell (PC3) lines. Hemolytic assay exhibits insignificant hemolysis of human erythrocyte cells at the minimum inhibitory concentration (MIC) of these tested bacteria. In this regard the present invention of 4-hydroxycoumarin based antimicrobial and noncytotoxic 4HBD molecule holds future promise in the development of new antibiotics.

Investigation on the interaction of Rutin with serum albumins: Insights from spectroscopic and molecular docking techniques

The binding interaction of Rutin, a flavonoid, with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), were investigated using different spectroscopic techniques, such as fluorescence, time-resolved single photon counting (TCSPC) and circular dichroism (CD) spectroscopy as well as molecular docking method. The emission studies revealed that the fluorescence quenching of BSA/HSA by Rutin occurred through a simultaneous static and dynamic quenching process, and we have evaluated both the quenching constants individually. The binding constants of Rutin-BSA and Rutin-HSA system were found to be 2.14 × 106 M−1 and 2.36 × 106 M−1 at 298 K respectively, which were quite high. Further, influence of some biologically significant metal ions (Ca2+, Zn2+ and Mg2+) on binding of Rutin to BSA and HSA were also investigated. Thermodynamic parameters justified the involvement of hydrogen bonding and weak van der Waals forces in the interaction of Rutin with both BSA and HSA. Further a site-marker competitive experiment was performed to evaluate Rutin binding site in the albumins. Additionally, the CD spectra of BSA and HSA revealed that the secondary structure of the proteins was perturbed in the presence of Rutin. Finally protein–ligand docking studies have also been performed to determine the probable location of the ligand molecule.

The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.Support or Funding InformationArnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Near shot-noise limited time-resolved circular dichroism pump-probe spectrometer.

We describe an optical near shot-noise limited time-resolved circular dichroism (TRCD) pump-probe spectrometer capable of reliably measuring circular dichroism signals in the order of μdeg with nanosecond time resolution. Such sensitivity is achieved through a modification of existing TRCD designs and introduction of a new data processing protocol that eliminates approximations that have caused substantial nonlinearities in past measurements and allows the measurement of absorption and circular dichroism transients simultaneously with a single pump pulse. The exceptional signal-to-noise ratio of the described setup makes the TRCD technique applicable to a large range of non-biological and biological systems. The spectrometer was used to record, for the first time, weak TRCD kinetics associated with the triplet state energy transfer in the photosynthetic Fenna-Matthews-Olson antenna pigment-protein complex.

Time and Spatially Resolved Hard X-Ray MCD Measurement on a Co/Pt Multilayer Dot Excited by Pulsed RF Field

Understanding of magnetization behavior in magnetic nanostructures is a key issue for future magnetic/spintronic devices. Time-resolved X-ray magnetic circular dichroism (XMCD) is a powerful measurement technique with potential of nanometer spatial resolution, picosecond time resolution, and element selectivity. We have carried out time and spatially resolved XMCD microscopy measurement on a Co/Pt multilayer dot by detecting XMCD at the Pt $L_{3}$ edge at BL39XU of SPring-8 using hard X-ray. Transient magnetic response of Co/Pt multilayer dots with diameter of $2.6~\mu \text{m}$ was investigated against pulsed RF field with frequency around 2.5 GHz and the maximum amplitude of about 400 Oe. By synchronizing excitation RF field pulse with X-ray pulses, the growth of magnetization precession of the induced Pt magnetic moment in the dot was clearly observed with sub-100 ps time resolution and submicrometer spatial resolution. The developed measurement setup can be applicable for a wide range of studies for time-resolved hard X-ray experiments.

Probing the stereoselective interaction of ofloxacin enantiomers with corresponding monoclonal antibodies by multiple spectrometry

Although stereoselective antibody has immense potential in chiral compounds detection and separation, the interaction traits between stereoselective antibody and the corresponding antigenic enantiomers are not yet fully exploited. In this study, the stereospecific interactions between ofloxacin isomers and corresponding monoclonal antibodies (McAb-WR1 and McAb-MS1) were investigated using time-resolved fluorescence, steady-state fluorescence, and circular dichroism (CD) spectroscopic methods. The chiral recognition discrepancies of antibodies with ofloxacin isomers were reflected through binding constant, number of binding sites, driving forces and conformational changes. The major interacting forces of McAb-WR1 and McAb-MS1 chiral interaction systems were hydrophobic force and van der Waals forces joined up with hydrogen bonds, respectively. Synchronous fluorescence spectra and CD spectra results showed that the disturbing of tyrosine and tryptophan micro-environments were so slightly that no obvious secondary structure changes were found during the chiral hapten binding. Clarification of stereospecific interaction of antibody will facilitate the application of immunoassay to analyze chiral contaminants in food and other areas.

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy.

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β4 homotetramer) and, therefore, are associated with the α-β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.

Spin currents during ultrafast demagnetization of ferromagnetic bilayers

Ultrafast spin currents induced by femtosecond laser excitation of ferromagnetic metals have been found to contribute to sub-picosecond demagnetization, and to cause a transient enhancement of the magnetization of the bottom Fe layer in a Ni/Ru/Fe layered structure. We analyze the ultrafast magnetization dynamics in such layered structures by element- and femtosecond time-resolved x-ray magnetic circular dichroism, for different Ni and Fe layer thicknesses, Ru and Ta interlayers, and by varying the pump laser fluence. While we do not observe the transient enhancement of the magnetization in Ni/Ru/Fe discovered previously, we do find a reduced demagnetization of the Fe layer compared to a Ni/Ta/Fe layered structure. In the latter, the spin-scattering Ta layer suppresses spin currents from the Ni layer into Fe, consistent with previous results. Any spin current arriving in the lower Fe layer will counteract other, local demagnetization mechanisms such as phonon-mediated spin-flip scattering. We find by increasing the Ni and Fe layer thicknesses in Ni/Ru/Fe a decreasing effect of spin currents on the buried Fe layer, consistent with a mean free path of the laser-induced spin currents of just a few nm. Our results suggest that in order to utilize ultrafast spin currents in an efficient manner, the sample design has to be optimized with these considerations in mind, and further studies clarifying the role of interfaces in the employed layered structures are needed.

Probing the binding properties of dicyandiamide with pepsin by spectroscopy and docking methods

Dicyandiamide (DCD), considered to be a nitrification inhibitor, poses threat to human’s health with exposure from milk, infant formula and other food products. In this work, DCD was investigated for its binding reaction with pepsin using spectroscopy and docking methods. Fluorescence experiments indicated DCD quenched the fluorescence of pepsin through a static process. Thermodynamic analysis of the binding data (ΔH0 = −21.72 kJ mol−1 and ΔS0 = 17.61 J mol−1 K−1) suggested the involvement of hydrophobic and hydrogen bonding in the complex formation. The pepsin interacted with DCD at a hydrophobic cavity, leading to a conformational changes in the pepsin, as revealed from UV–vis absorption, Fourier transform infrared, the time-resolved fluorescence, three-dimensional fluorescence and circular dichroism spectral results.

Pump-probe experiments at the TEMPO beamline using the low-α operation mode of Synchrotron SOLEIL.

The SOLEIL synchrotron radiation source is regularly operated in special filling modes dedicated to pump-probe experiments. Among others, the low-α mode operation is characterized by shorter pulse duration and represents the natural bridge between 50 ps synchrotron pulses and femtosecond experiments. Here, the capabilities in low-α mode of the experimental set-ups developed at the TEMPO beamline to perform pump-probe experiments with soft X-rays based on photoelectron or photon detection are presented. A 282 kHz repetition-rate femtosecond laser is synchronized with the synchrotron radiation time structure to induce fast electronic and/or magnetic excitations. Detection is performed using a two-dimensional space resolution plus time resolution detector based on microchannel plates equipped with a delay line. Results of time-resolved photoelectron spectroscopy, circular dichroism and magnetic scattering experiments are reported, and their respective advantages and limitations in the framework of high-time-resolution pump-probe experiments compared and discussed.

Capturing ultrafast magnetic dynamics by time-resolved soft x-ray magnetic circular dichroism

Experiments of time-resolved x-ray magnetic circular dichroism (Tr-XMCD) and resonant x-ray scattering at a beamline BL07LSU in SPring-8 with a time-resolution of under 50 ps are presented. A micro-channel plate is utilized for the Tr-XMCD measurements at nearly normal incidence in both the partial electron and total fluorescence yield (PEY and TFY) modes at the L2,3 absorption edges of the 3d transition-metals in the soft x-ray region. The ultrafast photo-induced demagnetization within 50 ps is observed on the dynamics of a magnetic material of FePt thin films, having a distinct threshold of the photon density. The spectrum in the PEY mode is less-distorted at both the L2,3 edges compared with that in the TFY mode and has the potential to apply the sum rule analysis for XMCD spectra in pump-probed experiments.

Interaction of Different Divalent Metal Ions with Lipid Bilayer: Impact on the Encapsulation of Doxorubicin by Lipid Bilayer and Lipoplex Mediated Deintercalation.

In this article, we investigate the influence of different metal ions (Ca2+, Mg2+, and Zn2+) on binding of an anticancer drug doxorubicin (DOX) to DMPC bilayer and lipoplex mediated deintercalation of DOX from DOX-DNA complex. Our study reveals that lipid bilayer in the presence of different metal ions displays much higher binding affinity toward DOX than bare lipid bilayer does. Further, this affinity for a particular metal ion increases linearly with metal ion concentration. The steady state and time-resolved fluorescence studies reveal that binding of DOX with lipid bilayer in the presence of different metal ions varies in the order of Ca2+> Mg2+> Zn2+. The rotational relaxation of DOX in the presence of different metal ions takes place in the same order. We explain these phenomena in the light of alteration of the physical properties brought about by metal ions. Moreover, we find that binding pattern of metal ions with lipid head groups influences the intake of DOX in lipid bilayer. We exploit the binding of DOX with bilayer to study the deintercalation of DOX from DOX-DNA complex. We observe that with increase in metal ion concentration the deintercalation increases. Among all metal ions, Ca2+ appears to be most effective in deintercalation compared to other metal ions. The time-resolved fluorescence anisotropy and circular dichroism measurements indicate that in the presence of Ca2+, lipid bilayer offer strongest interaction with DNA while the same is weakest for Zn2+. This explains the highest percentage of deintercalation of DOX from drug-DNA complex in the presence of Ca2+. Overall the present study demonstrates a new strategy that binding of drug molecules with lipid bilayer and deintercalation of the same from drug-DNA complex can be tuned by modulation of lipid bilayer with different metal ions and their concentration.

Photoinduced molecular chirality probed by ultrafast resonant X-ray spectroscopy.

Recently developed circularly polarized X-ray light sources can probe the ultrafast chiral electronic and nuclear dynamics through spatially localized resonant core transitions. We present simulations of time-resolved circular dichroism signals given by the difference of left and right circularly polarized X-ray probe transmission following an excitation by a circularly polarized optical pump with the variable time delay. Application is made to formamide which is achiral in the ground state and assumes two chiral geometries upon optical excitation to the first valence excited state. Probes resonant with various K-edges (C, N, and O) provide different local windows onto the parity breaking geometry change thus revealing the enantiomer asymmetry.

Relaxation Dynamics in Photoexcited Chiral Molecules Studied by Time-Resolved Photoelectron Circular Dichroism: Toward Chiral Femtochemistry

Unravelling the main initial dynamics responsible for chiral recognition is a key step in the understanding of many biological processes. However, this challenging task requires a sensitive enantiospecific probe to investigate molecular dynamics on their natural femtosecond time scale. Here we show that, in the gas phase, the ultrafast relaxation dynamics of photoexcited chiral molecules can be tracked by recording time-resolved photoelectron circular dichroism (TR-PECD) resulting from the photoionization by a circularly polarized probe pulse. A large forward–backward asymmetry along the probe propagation axis is observed in the photoelectron angular distribution. Its evolution with pump–probe delay reveals ultrafast dynamics that are inaccessible in the angle-integrated photoelectron spectrum or via the usual electron emission anisotropy parameter (β). PECD, which originates from the electron scattering in the chiral molecular potential, appears as a new sensitive observable for ultrafast molecular dynamics in chiral systems.

Lipoplex-Mediated Deintercalation of Doxorubicin from Calf Thymus DNA-Doxorubicin Complex.

In this paper, we report the lipoplex-mediated deintercalation of anticancer drug doxorubicin (DOX) from the DOX-DNA complex under controlled experimental conditions. We used three zwitterionic liposomes, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), which are widely different in their phase transition temperatures to form a lipoplex with calf thymus DNA in the presence of Ca(2+) ions. The study revealed that DPPC being in sol-gel phase was more effective in releasing the drug from the DOX-DNA complex compared with liposomes that remain in liquid crystalline phase (DMPC and POPC). The higher extent of drug release in the case of DPPC liposomes was attributed to the stronger lipoplex formation with DNA as compared with that of other liposomes. Owing to the relatively smaller head group area, the DPPC liposomes in their sol-gel phase can absorb a larger number of Ca(2+) ions and hence offer a strong electrostatic interaction with DNA. This interaction was confirmed by time-resolved anisotropy and circular dichroism spectroscopy. Apart from the electrostatic interaction, the possible hydrophobic interaction between the liposomes and DNA was also taken into account for the observed deintercalation. The successful uptake of drug molecules by liposomes from the drug-DNA complex in the post-release period was also confirmed using confocal laser scanning microscopy (CLSM).