Mechanism of isomerization of rhodopsin studied by use of 11-cis-locked rhodopsin analogues excited with a picosecond laser pulse

Primary photochemical behaviors of cattle rhodopsin analogues (Rh5 and Rh7) having cyclopenta- and cycloheptatrienylidene 11-cis-locked retinals (Ret5 and Ret7, respectively) were studied by excitation with a picosecond laser pulse (wavelength 532 nm; duration 21 ps). Picosecond absorption and fluorescence measurements of Rh5 showed formation of only a long-lived excited singlet state (tau l/e = 85 ps). The excited state of the retinal analogue having a five-membered ring was stabilized in protein (Rh5) more than in solvent (protonated Schiff base of Ret5; PSB5). Excitation of Rh7 produced two ground-state photoproducts, Rh7 (580) and Rh7 (630). According to the analysis of photon density dependency, Rh7 (580) was a single-photon product of Rh7, while Rh7 (630) was the photoproduct of Rh7 (580). Fluorescence emitted from a seven-membered ring system like Rh7 or a protonated Schiff base of Ret7 (PSB7) was weaker than that in a corresponding five-membered ring system, especially in protein (Rh7). The difference in photoreaction between Rh5 and Rh7 may originate from the difference in fixation of the 11-cis form. On the basis of the spectral and kinetic similarities between Rh7 (580) and photorhodopsin, a precursor of bathorhodopsin, it was proposed that both have twisted all-trans chromophores in the way of the isomerization. The protein moiety of rhodopsin which fixes the chromophore at both ends seems to accelerate the rotation of the C11-C12 double bond and to prevent it from going through relaxation processes other than the isomerization. This may be a plausible reason why rhodopsin has a large quantum yield (0.67).     

Emission of terahertz waves in the interaction of a laser pulse with clusters

A theory of generation of terahertz radiation in the interaction of a femtosecond laser pulse with a spherical cluster is developed for the case in which the density of free electrons in the cluster plasma exceeds the critical value. The spectral, angular, and energy characteristics of the emitted terahertz radiation are investigated, as well as its spatiotemporal structure. It is shown that the directional pattern of radiation has a quadrupole structure and that the emission spectrum has a broad maximum at a frequency nearly equal to the reciprocal of the laser pulse duration. It is found that the total radiated energy depends strongly on the cluster size. Analysis of the spatiotemporal profile of the terahertz signal shows that it has a femtosecond duration and contains only two oscillation cycles.     

Red chlorophyll excitation dynamics in Arthrospira platensis photosystem I trimeric complexes as studied by femtosecond transient absorption spectroscopy

Femtosecond absorption spectroscopy was applied to study for the first time excitation dynamics in isolated photosystem I trimers from Arthrospira platensis, which display extremely long-wavelength absorption peaks. Pump–probe spectra observed at 77 K in the timescale of dozens of picoseconds upon 70-fs excitation revealed two maxima near 710 and 730 nm, which correspond to red chlorophyll forms. Bleaching at 680 nm developed in ∼200 fs, whereas the bleaching kinetics at 710 and 730 nm exhibited two components with time constants of 1 and 5.5 ps. Comparison of the kinetics of bleaching development at 710 nm and 730 nm with that of bleaching decay at 680 nm indicated that both long-wavelength forms of trimers are populated mainly via direct energy transfer from bulk chlorophyll.     

Weibel instability in the field of a short laser pulse

The growth rate of Weibel instability in a plasma interacting with a high-frequency pulse with a duration less or comparable with the electron mean free time is determined. The growth rate is shown to decrease with decreasing pulse duration. It is found that instability can develop after the short pulse is switched off and the generated magnetic field no longer affects electron motion in the high-frequency field.     

Measuring fast dynamics in solutions and cells with a laser scanning microscope

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.     

Thomson scattering in a plasma created by a short intense laser pulse

Thomson scattering spectra from a plasma created through ionization of a gas consisting of multielectron atoms by a laser pulse with an intensity of about 10¹⁶ W/cm² or higher and with a duration τ_imp≤100 fs are studied theoretically with allowance for electron groups with different temperatures.     

Real-time structural investigation of a lipid bilayer during its interaction with melittin using sum frequency generation vibrational spectroscopy

Interactions between membrane bilayers and peptides/proteins are ubiquitous throughout a cell. To determine the structure of membrane bilayers and the associated peptides/proteins, model systems such as supported lipid bilayers are often used. It has been difficult to directly investigate the interactions between a single membrane bilayer and peptides/proteins without exogenous labeling. In this work we demonstrate that sum frequency generation vibrational spectroscopy can be employed to study the interactions between peptides/proteins and a single lipid bilayer in real time, in situ, and without exogenous labeling. Using melittin and a dipalmitoyl phosphatidylglycerol bilayer as a model system, we monitored the C-H and C-D stretching signals from isotopically symmetric or asymmetric dipalmitoyl phosphatidylglycerol bilayers during their interaction with melittin. It has been found that the extent and kinetics of bilayer perturbation induced by melittin are very sensitive to melittin concentration. Such concentration dependence is correlated to melittin's mode of action. Melittin is found to function via the early and late stage of the carpet model at low and high concentrations, respectively, whereas the toroidal model is probable at intermediate concentrations. This research illustrates the potential of sum frequency generation as a biophysical technique to monitor individual leaflet structure of lipid bilayers in real time during their interactions with biomolecules.     

Human microflora in the norm and in pathology studied with laser fluorescence

On the basis of experimental and clinical data the use of laser fluorescence for indication of microflora in normal and pathological states has been substantiated. Standardized intensity characteristics of fluorescence integrally reflect the presence and activity of the total microflora and its changes in dysbiosis and pyo-inflammatory diseases. The possibility of its use in clinical practice is shown.     

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (ΔF ∝ Osm⁻¹; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1–4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl₂, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of E_A=4.9 kcal mol⁻¹, in the absence of HgCl₂, and E_A=11.9 kcal mol⁻¹ in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg²⁺ ions and low activation energy.     

Spatio-temporal dynamics of chlorophyll in the open Baltic Sea

Five charts of the chlorophyll and hydrographic fields by verticalprofiles of in situ fluorescence and CTD were made on a stationarygrid of 20 ? 25 nautical miles with a 5-mile spacing in theopen Baltic Sea. Both chlorophyll levels and variability weremaximal close to the spring bloom. High chlorophyll levels insummer are sustained by recurrent nutrient injections from thedeep saline layer. Two of the surveys showed close couplingbetween the coarse-scale (10 km) chlorophyll distribution andthe hydrographic structure determining the intensity of nutrienttransfer. Vigorous advection, stirring and current shear, associatedwith a strong mesoscale eddy, probably dominated the chlorophyllpattern on three surveys. The upward velocities in the cycloniceddy resulted in accumulation of phytoplankton in the aphoticzone. Intensive heat input from the surface caused a suddensinking of the phytoplankton and the formation of a pronouncedsub-surface chlorophyll maximum.     

Emission of low-frequency electromagnetic waves by a short laser pulse propagating in a plasma with density fluctuations

It is shown that a short laser pulse propagating in a plasma with electron density fluctuations can emit electromagnetic waves with frequencies much lower than the laser carrier frequency. Emissions with frequencies close to the plasma frequency and the doubled plasma frequency in a nonisothermal plasma, as well as emission generated in a turbulent plasma, are examined. The effects in question are related to the transformation of the laser pulse wakefield into electromagnetic radiation by electron density fluctuations. The phenomenon under study opens new possibilities for diagnostics of both plasma fields excited by laser pulses and electron density fluctuations in a plasma.     

The structure of a membrane-spanning polypeptide studied by molecular dynamics

We have performed a molecular dynamics simulation of a 46-residue segment of glycophorin which includes the hydrophobic membrane-spanning region of this protein. The presence of a membrane and of water is taken into account in a continuum approximation which makes use of phenomenological hydrophobic energies. The initial -helical conformation and the membrane incorporation of the hydrophobic segment remain stable for the length of the simulation which is 100 ps. Moreover, when the hydrophobic segment is partially shifted out of the membrane, it moves back into the membrane. Superimposed on these deterministic effects one also observes thermal fluctuations in the form of bending and tilting of the membrane-spanning helix.     

Excitation of Langmuir oscillations by a laser pulse in a semi-infinite dense plasma

A study is made of the nonlinear mechanism for the excitation of Langmuir waves in a dense plasma by an intense laser pulse with the frequency ω = ω_p/2 (where ω_p is the electron plasma frequency).     

Crater formation in a target under the action of a high-power laser pulse

The formation of craters in targets of various materials under the action of a high-power neodymium-laser pulse at radiation intensities from 10¹⁰ to 10¹⁴ W/cm² was studied experimentally and theoretically. The interaction between the laser beam and solid targets is investigated to determine the efficiency of the ablation loading of various materials and the transformation of the laser energy into the energy of a shock wave.     

Mechanisms of selectivity in channels and enzymes studied with interactive molecular dynamics

Interactive molecular dynamics, a new modeling tool for rapid investigation of the physical mechanisms of biological processes at the atomic level, is applied to study selectivity and regulation of the membrane channel protein GlpF and the enzyme glycerol kinase. These proteins facilitate the first two steps of Escherichia coli glycerol metabolism. Despite their different function and architecture the proteins are found to employ common mechanisms for substrate selectivity: an induced geometrical fit by structurally homologous binding sites and an induced rapid dipole moment reversal. Competition for hydrogen bonding sites with water in both proteins is critical for substrate motion. In glycerol kinase, it is shown that the proposed domain motion prevents competition with water, in turn regulating the binding of glycerol.