Ultrafast dynamics of myoglobin probed by time-resolved resonance Raman spectroscopy

Recent experimental work carried out in this laboratory on the ultrafast dynamics of myoglobin (Mb) is summarized with a stress on structural and vibrational energy relaxation. Studies on the structural relaxation of Mb following CO photolysis revealed that the structural change of heme itself, caused by CO photodissociation, is completed within the instrumental response time of the time-resolved resonance Raman apparatus used (approximately 2 ps). In contrast, changes in the intensity and frequency of the iron-histidine (Fe-His) stretching mode upon dissociation of the trans ligand were found to occur in the picosecond regime. The Fe-His band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous, in contrast with that observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. The band position of the Fe-His stretching mode changed with a time constant of about 100 ps, indicating that tertiary structural changes of the protein occurred in a 100-ps range. Temporal changes of the anti-Stokes Raman intensity of the v4 and v7 bands demonstrated immediate generation of vibrationally excited heme upon the photodissociation and decay of the excited populations, whose time constants were 1.1 +/- 0.6 and 1.9 +/- 0.6 ps, respectively. In addition, the development of the time-resolved resonance Raman apparatus and prospects in this research field are described.     

Intracellular localization of meso-tetraphenylporphine tetrasulphonate probed by time-resolved and microscopic fluorescence spectroscopy.

The effects of solvent pH on spectral properties and fluorescence decay kinetics were investigated in order to characterize the microenvironment of meso-tetraphenylporphine tetrasulphonate (TPPS4) taken up by cells. Steady-state absorption and fluorescence spectra of TPPS4 in buffer solutions of different pH were used to identify a ring protonated species at pH less than or equal to 4. This dictation could also be distinguished from the unprotonated form by its altered fluorescence decay time (3.5 vs. 11.4 ns). In addition, time-resolved spectroscopy gave some evidence of a monocationic species existing at pH 6-9. This was concluded from the occurrence of another component with a decay time of 5 ns. Measurements of the spectral and kinetic properties of the fluorescence emission of single epithelial cells (RR1022) incubated with TPPS4 indicated that the sensitizer was mainly localized in a microenvironment with a pH of 5, a value which occurs intracellularly only within lysosomes. Cells kept in the dark exhibited the characteristic spectra of both the dication and the neutral form. The fluorescence decay showed two components with decay times of 2.6 ns and 10.6 ns. Irradiation of the cells changed the decay times to 4.6 ns and 13.4 ns and the dication fluorescence emission peak vanished, which is in accordance with the results obtained from buffer solutions at pH greater than or equal to 6. Therefore, we deduce that the photodynamic action leads to a rupture of the lysosomes and that the sensitizer is released into the surrounding cytoplasm.     

Laser-induced potential jump at the electrochemical interface probed by picosecond time-resolved surface-enhanced infrared absorption spectroscopy

Picosecond time-resolved surface-enhanced infrared absorption spectroscopy (SEIRAS) has been used for the first time to examine the potential jump at the electrochemical interface induced by a visible pulse irradiation. The potential dependent shift of the C-O stretching vibration of CO adsorbed on a Pt electrode was utilized to monitor the potential jump. A 6-cm(-1) red-shift was observed with a time delay of approximately 200 ps with respect to a visible pump-pulse irradiation (532 nm, 35 ps duration, 3 mJ cm(-2)). The observed red-shift is ascribed to the heating of the in-plane frustrated translational mode of CO and the negative shift of potential. These two contributions can be separated with the aid of the transient of the background reflectivity of the electrode surface. The heating of water layers near the surface is mainly responsible for the potential jump through the orientation change of water molecules. This method is promising as a tool to examine ultrafast electrode dynamics.     

Nanoscale organization of ionic liquids and their interaction with peptides probed by C NMR spectroscopy

An NMR study of 10 l-alanine- and l-valine-containing peptides was carried out in the native [C₂MIM][Cl], [C₄MIM][Cl], [C₆MIM][Cl], [C₄MIM][BF₄], [C₄MIM][PF₆], and [C₄Py][BF₄] ionic liquid media. A unique high sensitivity of the ionic liquid system to the nature of peptide and ability to tune solvent–solute interactions were observed in contrast to regular organic solvents. The l-valine peptides can be selectively dissolved in [C₄MIM][Cl] and [C₆MIM][Cl], whereas their solubility in [C₂MIM][Cl] and other ionic liquids was dramatically lower. In spite of structural similarity between the amino acids, a distinct behavior was observed for the l-alanine peptides. Solvent–solute interactions with an ionic liquid impose significant changes, and NMR spectroscopy is a useful probe for the molecular-level and nanoscale organization of the studied systems. An even/odd effect of the number of amino acids in the peptide on molecular interactions in ionic liquids was observed. Enhancement of chemical properties of peptides in ionic liquids and application of ionic liquids in the separation of peptides are the areas of practical interest in the studied systems.     

Proteins in action monitored by time-resolved FTIR spectroscopy.

In the post genome era proteins coming into the focus of life sciences. X-ray structure analysis and NMR spectroscopy are established methods to determine the geometry of proteins. In order to determine the molecular reaction mechanism of proteins, time-resolved FTIR (trFTIR) difference spectroscopy emerges as a valuable tool. In this Minireview we describe the trFTIR difference spectroscopy and show its application on the light-driven proton pump bacteriorhodopsin (bR), the photosynthetic reaction center and the GTPase Ras, which is crucial in signal transduction. The main principles of the technique are presented, including a summary of triggering techniques, scan modes and analysis.     

Identification of peroxo groups in supramolecular layered structures as probed by Raman spectroscopy

Peroxide-containing supramolecular structures prepared by reacting lithium aluminum layered double hydroxides (Li-Al LDHs) with concentrated hydrogen peroxide solutions were characterized by Raman spectroscopy. These compounds were formulated as [LiAl₂(OH)₆](OH) · H₂O₂ · H₂O(I) and [LiAl₂(OH)₆](OOH) · H₂O₂ · H₂O(II). The frequencies 830 and 849 cm⁻¹ in the spectra of compounds I and II were assigned to O—C stretching vibrations in two nonequivalent peroxo groups. The band at 866 cm⁻¹ in compound II was assigned to O—O vibrations in the hydroperoxo group (OOH)⁻. Proceeding from calculated strength factors, we inferred that the O—O bond in the hydroperoxo group of compound II is stronger than in the H₂O₂ solvating group. Original Russian Text ? T.A. Tripol’skaya, I.V. Pokhabova, P.V. Prikhodchenko, G.P. Pilipenko, E.A. Legurova, N.A. Chumaevskii, 2009, published in Zhurnal Neorganicheskoi Khimii, 2009, Vol. 54, No. 3, pp. 513–515.     

Elucidating interactions of ionic liquids with polymer films using confocal Raman spectroscopy

We report on the molecular interactions between room-temperature ionic liquids (RTILs) and Nafion and PDMS membranes, proving that in contact with these polymers RTILs behave like electrolytes rather than solvents.     

Structure of water at ionic liquid/Ag interface probed by surface enhanced Raman spectroscopy

The potential-dependent adsorption behavior of water and ionic liquid was probed by surface-enhanced Raman spectroscopy (SERS) at the Ag electrode surface in the ionic liquids containing water with different concentrations.The configuration of water at the ionic liquid/electrode interface and the relationship between the potential of zero charge (pzc) and the molar fraction of water were deduced through the changes in the vibrational frequency of OH stretching mode.A small Stark effect value was determined ...     

含水离子液体/金属界面结构的SERS研究

利用表面增强拉曼光谱(SERS)研究了不同含水量下离子液体及水分子在银电极上随电位变化吸附方式的改变,通过水的O-H伸缩振动谱峰频率变化特征,详细探究了水在离子液体/电极界面上的存在形式及作用方式以及体系零电荷电位与水含量的关系.水含量较低时O-H伸缩振动的Stark系数值较低,随水含量的增加O-H伸缩振动的谱峰位置逐渐向高波数方向移动,同时O-H伸缩振动的Stark系数也逐渐增大,1molL-1[BMIM]Br水溶液中达到76cm-1V-1,且体系的零电荷电位正移,这些差异与水在离子液体中所形成氢键的程度及水分子的存在形式密切相关,在水的含量较低时水与离子液体阳离子通过氢键作用而存在于界面层中,当水的含量增加时,水分子间氢键的作用增强,水与电极表面直接作用的可能性增大.     

Microscopic structures of ionic liquids 1-ethyl-3-methylimidazolium tetrafluoroborate in water probed by the relative chemical shift

The relative chemical shifts (Δδ) were put forward to investigate the microscopic structure of 1-ethyl-3-methyl-imidazolium tetrafluoroborate (EmimBF₄) during the dilution process with water. The concentration-dependent Δδ _{(C2)H–(C4)H}, Δδ _{(C2)H–(C5)H} and Δδ _{(C4)H–(C5)H} were analyzed. The results reveal that the variations of the microscopic structures of three aromatic protons are inconsistent. The strength of the H-bond between water and three aromatic protons follows the order: (C2)H…O > (C4)H…O > (C5)H…O. The concentration-dependent Δδ _{(C6)H–(C7)H} and Δδ _{(C6)H–(C8)H} indicate the formation of the H-bonds of (C_alkyl)H…O is impossible, and more water is located around (C6)H than around (C7)H or (C8)H. The concentration-dependent Δδ _{(C2)H–(C4)H} and Δδ _{(C2)H–(C5)H} both increase rapidly when x _water > 0.9 or so, suggesting the ionic pairs of EmimBF₄ are dissociated rapidly. The turning points of concentration-dependent Δδ _{(C2)H–(C4)H} and Δδ _{(C2)H–(C5)H} indicate that some physical properties of the EmimBF₄/water mixtures also change at the corresponding concentration point. The microscopic structures of EmimBF₄ in water could be clearly detected by the relative chemical shifts.     

Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy

Phototropins are light-activated kinases from plants that utilize light-oxygen-voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an alpha-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.     

Tail aggregation and domain diffusion in ionic liquids

An extended multiscale coarse-graining model for ionic liquids is used to investigate the liquid crystal-like phase in certain ionic liquids. The tail groups of the cations with a sufficient side-chain length are found to aggregate, forming spatially heterogeneous domains, due to the competition between the electrostatic interactions between the charged head groups and the anions and the collective short-range interactions between the neutral tail groups. With a sufficiently long alkyl chain at a low enough temperature, the tail domains remain relatively stable, despite the diffusion of individual ions in the liquid phase. With increasing temperature, the average tail domains begin to diffuse, while beyond a transition temperature, their average density has an almost uniform distribution, although the tail groups still form instantaneous domains.     

Dynamics of CO in mesoporous silica monitored by time-resolved step-scan and rapid-scan FT-IR spectroscopy

Carbon monoxide molecules generated in the channels of mesoporous MCM-41 silica sieve from a precursor (diphenyl cyclopropenone) by photodissociation with a nanosecond laser pulse were monitored by time-resolved Fourier transform infrared (FTIR) spectroscopy using the step-scan and rapid-scan methods. A very broad absorption of CO is observed in the region 2200-2080 cm(-1) at room temperature that decays in a biphasic mode. Two-thirds of the band intensity decays on the hundreds of microsecond scale (lifetime 344 +/- 70 micros). The process represents the escape of the molecules through the mesopores into the surrounding gas phase, and a diffusion constant of 1.5 x 10(-9) m(2)/s is derived (assuming control by intra-MCM-41 particle diffusion). The broad profile of the absorption is attributed to contact of the random hopping CO with siloxane and silanol groups of the pore surface. Measurements using MCM-41 with the silanols partially capped by trimethyl silyl groups gave further insight into the nature of the IR band profile. These are the first observations on the diffusion behavior of carbon monoxide in a mesoporous material at room temperature. The residual carbon monoxide remains much longer in the pores and features distinct peaks at 2167 and 2105 cm(-1) characteristic for CO adsorbed on SiOH groups C end on and O end on, respectively. The bands decrease with time constants of 113 +/- 3 ms (2167 cm(-1)) and 155 +/- 15 ms (2105 cm(-1)) suggesting that CO in these sites is additionally trapped by surrounding diphenyl acetylene coproduct and/or precursor molecules.     

Local structure at the air/liquid interface of room-temperature ionic liquids probed by infrared-visible sum frequency generation vibrational spectroscopy: 1-alkyl-3-methylimidazolium tetrafluoroborates

The air/liquid interface of 1-alkyl-3-methylimidazolium tetrafluoroborates with the general formula [C(n)mim]BF(4) (n = 4-11) was studied using infrared-visible sum frequency generation (SFG) vibrational spectroscopy. The probability of the gauche defect per CH2-CH2 bond in the alkyl chain decreases as the number of carbon atoms in the alkyl chain increases. This observation suggests that the interaction between the alkyl chains is enhanced as the alkyl chain length becomes longer. The frequencies of the C-H stretching vibrational modes observed in the SFG spectra are higher than those of the corresponding peak positions observed in the infrared spectra of the bulk liquids. This shift is consistent with a structure in which the alkyl chain protrudes from the bulk liquid into the air. A local structure, which originates from the intermolecular interaction between the ionic liquid molecules, is proposed to explain these observations.     

Speciation of uranyl complexes in ionic liquids by optical spectroscopy

Uranyl complexes dissolved in room-temperature ionic liquids have diagnostic absorption and emission spectra which reflect the molecular symmetry and geometry. In particular, the characteristic vibrational fine structure of the absorption spectra allows identification of the molecular symmetry of a uranyl complex. The concept of speciation of uranyl complexes is illustrated for the hydrated uranyl ion, the tetrachloro complex [UO2Cl4]2-, the trinitrato complex [UO2(NO3)3]-, the triacetato complex [UO2(CH3COO)3]-, and the crown ether complex [UO2(18-crown-6)]2+ in imidazolium and pyrrolidinium bis(trifluoromethylsulfonyl)imide ionic liquids. The competition between 18-crown-6 and small inorganic ligands for coordination to the uranyl ion was investigated. The crystal structures of the hydrolysis product [(UO2)2(mu2-OH)2(H2O)6] [UO2Br4](18-crown-6)4 and imidazolium salt [C6mim]2[UO2Br4] are described.     

Containerless reaction monitoring in ionic liquids by means of Raman microspectroscopy

Reaction monitoring by Raman microspectroscopy in levitated room temperature ionic liquid (RTIL) droplets is reported. Due to their non-volatility, RTIL droplets are well-suited to act as wall-less microreactors. The droplets were produced by a piezoelectric flow-through microdispenser connected to an automated flow injection system and were levitated by an acoustic trap. Taking advantage of the flow system versatility, the sequence of reagents was easily changed to study a model organic reaction: the Knoevenagel condensation. The reaction was followed by Raman microspectrometry and the obtained spectra were analysed using multivariate curve resolution to retrieve the concentration profiles and pure spectra of reactants, intermediates and products involved in the reaction. In addition, information about solvation interactions was obtained by monitoring the desolvation process taking place when a volatile co-solvent evaporated from the droplet.     

Dynamics of phase transitions in a liquid crystal probed by Raman spectroscopy

Raman spectra of the Schiff 's base liquid crystalline compound 5O5, N-(4- n-pentyloxybenzylidene)-4'-n-pentylaniline, have been recorded as a function of temperature from 22 to 80°C in the 1140-1220 cm^-1 and 1550-1640 cm^-1 spectral regions. From careful deconvolution of the spectral features using Lorentzian profiles, precise values of peak positions, integrated intensities and linewidths of some selected Raman bands were obtained. The variations of the Raman spectral parameters with temperature are discussed in terms of changes in the molecular alignment and its effect on intra-/inter-molecular interactions at the Cr-G, G-SmF, SmF-SmC and SmA-N phase transitions. From a detailed study, it is inferred that the increased orientational/vibrational freedom of the alkyl chains, as well as the delocalization of the electron clouds, is responsible for the spectral anomalies at the Cr-G transition. Loss of positional ordering and twist around the -C₆H₄-N= bond takes place at the SmF-SmC transition. In the SmA-N transition, some evidence for the formation of cybotactic clusters was obtained.     

Structures of ionic liquids with different anions studied by infrared vibration spectroscopy

We investigated the structures of ionic liquids (1-butyl-3-methylimidazolium iodide [BMIM][I] and 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]) and their aqueous mixtures using attenuated total reflection (ATR) infrared absorption and Raman spectroscopy. The ATR spectrum in the CHx (x = 1, 2, 3) vibration region from 2800 to 3200 cm-1 was very different between [BMIM][BF4] and [BMIM][I] even though all the spectral features in this region were from the butyl chain and the imidazolium ring of the same cation. The spectrum did not change appreciably irrespective of the water concentration for [BMIM][BF4], whereas the spectrum from [BMIM][I] showed significant changes as the water concentration was increased, especially in CH-vibration modes from the imidazolium ring. For very diluted solutions both aqueous mixtures of [BMIM][I] and [BMIM][BF4] showed very similar spectra. Mixing of [BMIM][I] with heavy water (D2O) facilitated the isotopic exchange of the proton attached to the most acidic carbon of the imidazolium ring into deuterium from D2O, whereas even prolonged exposure to D2O did not induce any isotopic exchange for [BMIM][BF4]. Raman spectra around 600 cm(-1) indicative of the butyl chain conformation also changed differently as the water concentration was increased between [BMIM][I] and [BMIM][BF4]. These differences are considered to come from the variation in the position of the anion, where I- is expected to be closer to the C(2) hydrogen of the imidazolium cation and interacting more specifically as compared to BF(4-).