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The hydrogen/deuterium (H/D) exchange rate is an optimal measure for studying the structures and dynamics of hydrogen bonding systems, as it reflects the molecular contact environment and the strength of the hydrogen bonds. A method for rapid measurement of the H/D exchange reaction rates is required to examine the intermolecular environments of molecules in solutions. We developed a droplet collision atmospheric pressure infrared laser ablation mass spectrometry technique for this purpose. We obtained the H/D exchange reaction rate of cytochrome c in a methanol/H2O·D2O solution. We revealed that the first hydration shell of the cytochrome c molecule hinders the penetration of D2O to the surface of the molecule from the rates, which provides a novel method to investigate solution structures by a mass-spectrometric method. The droplet-collision mass spectrometry method developed in the present study can be extended to research on the molecular interactions in solutions, such as the mutual interactions of protein molecules, which are of importance in living cells.
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A liquid droplet can act as a microscale high-Q optical cavity via a whispering gallery mode, where light resonates with enhanced intensity. A collision of two droplets temporarily exhibits a unique morphology, which provides a more effective optical cavity than a single droplet. We investigated the mechanisms of cavity-enhanced fluorescence in colliding droplets of aqueous rhodamine 6G. Laser-excited spectra and fluorescence generation times were acquired. The fluorescence spectra had two peaks: one attributed to amplified spontaneous emission (ASE) and the other to lasing. The lasing generation time had a longer delay relative to that of ASE, which indicated that it required a longer propagation distance for the positive feedback. Overall, this provides a basis for the development of a highly efficient dye laser using colliding droplets.
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Stimulated Raman scattering (SRS) in a liquid droplet enhances the Raman scattering intensity through optical resonances. We have previously used a pendant drop at the tip of a capillary as the Raman-enhancing medium. In this study, we develop a new optical cavity for SRS measurement that consists of a spherical shell of organic liquid. This enables us to extend the applicability of the pendant-drop SRS method to liquids with low viscosity or low interfacial tension. This method is used to observe low-frequency modes of liquid benzene. The results indicate that the SRS emerges locally with respect to the drop size. The developed method extends the study of liquid structures based on vibrational spectroscopy.
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Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.
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ProteóliseRESUMO
Chemical reactions at the interface of reactive solutions are of importance for a full understanding of solution reactions. We investigate the chemical reaction induced by the collision of two droplets. The extent of the reaction is measured by analyzing spectra and images of the Raman scattered light emerging from the interface of the colliding droplets of H2SO4 and NH3 aqueous solutions. The obtained product concentration is lower than that expected from a simple diffusion model. The result indicates that a fresh interface is produced at the periphery of the mixing region of the colliding droplets. This study provides the basis to extend this method to measure rapid chemical reactions at the interface of colliding droplets.
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Biomolecules function in an aqueous environment. Elucidation of the hydration structures of biomolecules is hence important to understand their functions. Here, we investigated the hydration structure of lysozyme (Lys) in the gas phase by photodissociation and fluorescence spectroscopy in combination with droplet-beam laser ablation mass spectrometry. We found that water molecules are held inside and on the surface of the Lys molecule, and the hydration structure around the tryptophan residue changes by photoexcitation. This study provides a novel method to observe the hydration structures of large biomolecules at the molecular level.
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Muramidase/química , Sequência de Aminoácidos , Raios Infravermelhos , Terapia a Laser , Espectrometria de Massas , Processos Fotoquímicos , Conformação Proteica , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
Gas phase spectroscopy is a powerful tool for examining fundamental chemical structures and properties free from solvent molecules. We developed a gas-phase resonance Raman spectroscopy combined with IR-laser ablation of a droplet beam, which allowed us to elucidate local structures around chromophores in gas-phase proteins and DNAs. To demonstrate the potential of this approach, we applied this method to myoglobin, one of the heme proteins, and elucidated its structures in the gas phase and in aqueous solution. The experimental spectra are compared with calculated spectra of stable heme structures for the structural determination. These results show the oxidation/spin states of the Fe atom in myoglobin in the gas phase and were compared with the aqueous solution from the obtained resonant Raman spectra. The present method gives an important tool to investigate the gas-phase structure of large biomolecules.
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Raios Infravermelhos , Lasers , Mioglobina/química , Análise Espectral Raman/métodos , DNA/química , Gases , Heme/química , Oxirredução , Conformação ProteicaRESUMO
Low-frequency intermolecular motions are of importance for understanding the structure of molecular liquids, which can be elucidated by Raman spectroscopy. A liquid droplet provides a field where stimulated Raman scattering (SRS) readily proceeds. Our previous study showed that multiorder SRS is more effectively generated in colliding droplets than a single droplet. Here we report that the multiorder SRS generated in colliding benzene droplets included low-frequency bands of the liquid benzene, which is considered to represent a temporal state of liquid emerging in the course of the droplet collision. This method enables measurements of the low-frequency intermolecular motions of the novel liquid state emerging at the mixed region of the colliding droplet without suffering from background of Rayleigh scattering.
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An S-S bond containing maleimide-conjugated closo-dodecaborate (SSMID) was synthesised for identification of albumin binding sites. Three Lys residues, Lys221, Lys413 and Lys431, were identified as SSMID modification sites in addition to Cys34 in bovin serum albumin (BSA). Fluorescent-labelled MID-BSA was found to be accumulated in the cytosol of HeLa cells.
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Compostos de Boro/química , Dissulfetos/química , Maleimidas/química , Soroalbumina Bovina/química , Animais , Sítios de Ligação , Compostos de Boro/síntese química , Bovinos , Células HeLa , Humanos , Estrutura MolecularRESUMO
Nonlinear Raman spectroscopy is a versatile method to enhance the intensities of Raman scattering. It requires an intense light field that can be provided by a liquid droplet acting as a high-quality optical cavity. Here, colliding droplets were used as a novel optical cavity to enhance the intensity of Raman scattering. Specifically, multiorder stimulated Raman-scattered light was generated with significant intensity from colliding droplets of carbon tetrachloride (CCl4). The intensities of the Raman bands were analyzed with a simple theory that roughly reproduced the experimental spectrum. Overall, the method facilitates Raman spectroscopy of molecules in liquids because of its high sensitivity and resolution.
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X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.
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Dynamic properties of the metastable interface between two miscible solutions are investigated by the collision of two droplets. A clear interface is observed between the two colliding droplets. The interface moves in the colliding droplet toward the side where the original droplet has a lower surface tension. The interface is set to the middle of the colliding droplet by controlling the surface tension of the droplets to observe the chemical reactions at the droplet interface by cavity-enhanced Raman spectroscopy. This study provides a foundation for further research on the initial process of the chemical reactions of two miscible solutions.
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Mass transfer toward and across liquid surfaces is important for the interpretation of various interfacial phenomena, such as evaporation, adsorption, and mass accommodation, which have been investigated by the use of various methods. These studies, however, have focused on only one of the mass-transfer processes occurring at the surface. We investigate the surface concentration of alcohol molecules at aqueous droplet surfaces on the several-millisecond time scale using cavity-enhanced droplet Raman spectroscopy. A decrease and subsequent increase of the alcohol concentration are observed in a set of measurements, which arise from an evaporation and subsequent adsorption of the alcohol molecules at the surface. This facilitates an understanding of the surface kinetics of molecules at the liquid surfaces.
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Materials work in multicomponent forms. A wide range of compositions must be tested to obtain the optimum composition for a specific application. We propose optimization using a series of small levitated single particles. We describe a tandem-trap apparatus for merging liquid droplets and analyzing the merged droplets and/or dried particles that are produced from the merged droplets under levitation conditions. Droplet merging was confirmed by Raman spectroscopic studies of the levitated particles. The tandem-trap apparatus enables the synthesis of a particle and spectroscopic investigation of its properties. This provides a basis for future investigation of the properties of levitated single particles.
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A liquid-droplet injector has been developed that delivers pristine microcrystals to an X-ray irradiation area for conducting serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL). By finely tuning the pulsed liquid droplets in time and space, a high hit rate of the XFEL pulses to microcrystals in the droplets was achieved for measurements using 5 µm tetragonal lysozyme crystals, which produced 4265 indexable diffraction images in about 30 min. The structure was determined at a resolution of 2.3 Å from <0.3 mg of protein. With further improvements such as reduction of the droplet size, liquid droplets have considerable potential as a crystal carrier for SFX with low sample consumption.
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Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodosRESUMO
Gas-phase isolation of bovine serum albumin (BSA) from aqueous solutions is performed by IR laser ablation of a droplet beam. Multiply charged BSA ions (positive and negative) were produced by the IR laser irradiation onto a droplet beam of aqueous BSA solutions with various pH values prepared by addition of hydrochloric acid or sodium hydroxide to the solution. The isolation mechanism was discussed based on the charge state of the isolated BSA ions. A nanodroplet model explains the gas-phase charge distribution of the BSA ions. This study provides a fundamental basis for further studies of a wide variety of biomolecules in the gas phase isolated directly from solution.
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Raios Infravermelhos , Lasers , Soroalbumina Bovina/químicaRESUMO
Many important chemical reactions are induced by mixing two solutions. This paper presents a new way to measure rates of rapid chemical reactions induced by mixing two reactant solutions using a liquid-droplet collision. The coloring reaction of phenolphthalein (H2PP) by a reaction with NaOH is investigated kinetically. Liquid droplets of H2PP/ethanol and NaOH/H2O solutions are made to collide, which induces a reaction that transforms H2PP into a deprotonated form (PP(2-)). The concentration of PP(2-) is evaluated from the RGB values of pixels in the colored droplet images, and is measured as a function of the elapsed time from the collision. The obtained rate constant is (2.2 ± 0.7) × 10(3) M(-1) s(-1), which is the rate constant for the rate-determining step of the coloring reaction of H2PP. This method was shown to be applicable to determine rate constants of rapid chemical reactions between two solutions.
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Processes involved between colliding droplets were investigated using simultaneous analysis of spectra and images of Raman-scattered light emitted by irradiation with a pulsed laser. This enabled spatially and temporally resolved Raman spectra of the colliding droplets to be obtained. Colliding droplets of ethanol and water produce a characteristic protrusion from the contact point to the antipode of the water droplet in the course of interaction. From its Raman spectrum, the protrusion is seen to be composed of water. This result supports our surface-tension release model previously proposed to describe the mechanism of protrusion formation because the protrusion is the result of positive interference of a capillary wave propagating over the surface of the water droplet in this model.
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Molecules exhibit their intrinsic properties in their isolated forms. Investigations of isolated large biomolecules require an understanding of the detailed mechanisms for their emergence in the gas phase because these properties may depend on the isolation process. In this study, we apply droplet-beam laser-ablation mass spectrometry to isolate protein molecules in the gas phase by IR-laser ablation of aqueous protein solutions, and we discuss the isolation mechanism. Multiply charged hydrated lysozyme clusters were produced by irradiation of the IR laser onto a droplet beam of aqueous lysozyme solutions with various pH values prepared by addition of sodium hydroxide to the solution. The ions produced in the gas phase show significantly low abundance and have a lower number of charges on them than those in the aqueous solutions, which we explained using a nanodroplet model. This study gives quantitative support for the nanodroplet model, which will serve as a fundamental basis for further studies of biomolecules in the gas phase.
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Raios Infravermelhos , Lasers , Espectrometria de Massas , Muramidase/isolamento & purificação , Nanoestruturas/análise , Gases/química , Concentração de Íons de Hidrogênio , Íons/química , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Modelos Químicos , Muramidase/química , Tamanho da Partícula , SoluçõesRESUMO
We demonstrated that platinum nanoparticles dispersed in pure water were "soldered" by gold into higher-order structures such as "nanowebs". In practice, gold nanoparticles in water containing the platinum nanoparticles were melted by irradiation of a pulsed 532-nm laser, which excites selectively the surface plasmon band of the gold nanoparticles, and the melted gold nanoparticles solder the platinum nanoparticles together into the nanowebs. Optical absorption spectroscopy and electron microscopy together with electron probe microanalysis were employed to observe the structure of the nanowebs and to elucidate their formation dynamics.