György Váró

Kinetic and Thermodynamic Study of the Bacteriorhodopsin Photocycle over a Wide pH Range

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.

Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.

Regulation of cerebral endothelial cell morphology by extracellular calcium

Cerebral endothelial cells interconnected by tight and adherens junctions constitute the structural basis of the blood-brain barrier. Extracellular calcium ions have been reported to play an important role in the formation and maintenance of the junctional complex. However, little is known about the action of calcium depletion on the structural characteristics of cerebral endothelial cells. Using atomic force microscopy we analyzed the effect of calcium depletion and readdition on the shape and size of living brain endothelial cells. It was found that the removal of extracellular calcium from confluent cell cultures induced the dissociation of the cells from each other accompanied by an increase in their height. After readdition of calcium a gradual recovery was observed until total confluency was regained. We have also demonstrated that Rho-kinase plays an important role in the calcium-depletion-induced disassembly of endothelial tight and adherens junctions. The Rho-kinase inhibitor Y27632 could prevent the morphological changes induced by a lack of calcium as well. Our results suggest that calcium depletion induces Rho-kinase-dependent cytoskeletal changes that may be partly responsible for the disassembly of the junctional complex.

Direct Measurement of the Photoelectric Response Time of Bacteriorhodopsin via Electro-Optic Sampling

The photovoltaic signal associated with the primary photochemical event in an oriented bacteriorhodopsin film is measured by directly probing the electric field in the bacteriorhodopsin film using an ultrafast electro-optic sampling technique. The inherent response time is limited only by the laser pulse width of 500 fs, and permits a measurement of the photovoltage with a bandwidth of better than 350 GHz. All previous published studies have been carried out with bandwidths of 50 GHz or lower. We observe a charge buildup with an exponential formation time of 1.68 +/- 0.05 ps and an initial decay time of 31.7 ps. Deconvolution with a 500-fs Gaussian excitation pulse reduces the exponential formation time to 1.61 +/- 0.04 ps. The photovoltaic signal continues to rise for 4.5 ps after excitation, and the voltage profile corresponds well with the population dynamics of the K state. The origin of the fast photovoltage is assigned to the partial isomerization of the chromophore and the coupled motion of the Arg-82 residue during the primary event.

Electric Signals during the Bacteriorhodopsin Photocycle, Determined over a Wide pH Range

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.

Study of the photocycle and charge motions of the bacteriorhodopsin mutant D96N

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.

Terahertz radiation from bacteriorhodopsin reveals correlated primary electron and proton transfer processes

The kinetics of electrogenic events associated with the different steps of the light-induced proton pump of bacteriorhodopsin is well studied in a wide range of time scales by direct electric methods. However, the investigation of the fundamental primary charge translocation phenomena taking place in the functional energy conversion process of this protein, and in other biomolecular assemblies using light energy, has remained experimentally unfeasible because of the lack of proper detection technique operating in the 0.1- to 20-THz region. Here, we show that extending the concept of the familiar Hertzian dipole emission into the extreme spatial and temporal range of intramolecular polarization processes provides an alternative way to study ultrafast electrogenic events on naturally ordered biological systems. Applying a relatively simple experimental arrangement based on this idea, we were able to observe light-induced coherent terahertz radiation from bacteriorhodopsin with femtosecond time resolution. The detected terahertz signal was analyzed by numerical simulation in the framework of different models for the elementary polarization processes. It was found that the principal component of the terahertz emission can be well described by excited-state intramolecular electron transfer within the retinal chromophore. An additional slower process is attributed to the earliest phase of the proton pump, probably occurring by the redistribution of a H bond near the retinal. The correlated electron and proton translocation supports the concept, assigning a functional role to the light-induced sudden polarization in retinal proteins.

Picosecond and nanosecond components in bacteriorhodopsin light-induced electric response signal

Numerous investigations on the primary events of the bacteriorhodopsin photocycle indicate that the first steps of the energy transformation process take place in the 500 fs-5 ps region. These processes are known to be followed by others in the μs and ms regions. Recent observations indicate also the existence of nanosecond intermediate(s). Here we are reporting on direct measurements of the light-induced electric response signal of purple membrane carried out in the ps and ns regions. The laser flash-induced electric response of dried oriented purple membrane samples were detected by an ultrafast sampling oscilloscope. The measured kinetic curves were analyzed by exponential fitting and by a simulation-optimization method taking into account the time characteristics of the measuring setup. This analysis revealed a two phase real charge separation process. The first phase (tau = 21 ± 2 ps) coincides well with the overall bR-[unk] K transition. The second phase (tau = 6 ± 0.5 ns) can be correlated with the nanosecond optical transitions reported by several workers, or may be an optically silent charge movement inside the protein moiety or on the surface of the membrane.

Monolithically integrated bacteriorhodopsin-GaAs field-effect transistor photoreceiver.

We have applied the large photovoltage developed across a layer of selectively deposited bacteriorhodopsin to the gate terminal of a monolithically integrated GaAs-based modulation-doped field-effect transistor, which delivers an amplified photoinduced current signal. The integrated biophotoreceiver device exhibits a responsivity of 3.8 A/W. The optoelectronic integrated circuit is achieved by molecular-beam epitaxy of the field-effect transistors heterostructure, photolithography, and selective-area bacteriorhodopsin electrodeposition.