Time-resolved photoelectric and absorption signals from oriented purple membranes immobilized in gel

Kinetics of photoelectric and absorption response signals were measured on samples containing oriented purple membranes immobilized in polyacrylamide gel. The orientation and aggregation states of purple membranes remain constant independently of pH and ionic strength in such samples and the gel does not influence the protom pump. The ‘gel method’ described in this study enables direct investigation of proton pump of bacteriorhodopsin and a simultaneous measurement of absorption signals within a wide range of parameters of the solution surrounding purple membranes and offers possibilities for study of other types of membranes as well.     

Optical Shading Induces an In‐Plane Potential Gradient in a Semiartificial Photosynthetic System Bringing Photoelectric Synergy

Semiartificial photosynthetic systems have opened up new avenues for harvesting solar energy using natural photosynthetic materials in combination with synthetic components. This work reports a new, semiartificial system for solar energy conversion that synergistically combines photoreactions in a purple bacterial photosynthetic membrane with those in three types of transition metal–semiconductor Schottky junctions. A transparent film of a common transition metal interfaced with an n‐doped silicon semiconductor exhibits an in‐plane potential gradient when a light‐penetration variance is established on its surface by optical shading of photoabsorbing photosynthetic membranes. The in‐plane potential gradients (0.08–0.3 V) enable a directional charge transport between the synthetic and natural photoelectric systems, which is further enhanced in a device setting by a biocompatible thixotropic gel electrolyte that permeates the membrane multilayer, facilitating a strong and steady photoelectric current as high as 1.3 mA cm^?2, the highest achieved so far with any anoxygenic photosynthetic system.     

Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel.

When illuminated, oriented purple membranes isolated from Halobacterium halobium give a photoelectric effect. The frequency response of a photocurrent measuring system for purple membranes oriented and immobilized in a polyacrylamide gel is analyzed from DC to 100 MHz. The waveform of the photocurrent can depend on both the sample conditions (including bathing solution) and the measuring system (electrode and ammeter) at both the low and high frequency ends. In the DC-1 kHz range (millisecond signals), the apparent lifetime of the photocurrent component is distorted if the electrode is not platinized and if the conductivity of the bathing solution is not low. In the 1 kHz to 1 MHz range (microsecond signals), the frequency response is flat under most conditions. In the MHz range (nanosecond signals), the apparent lifetime of the photocurrent component will be distorted if the conductivity of the bathing solution is not high and if the input impedance of the ammeter is not low and constant throughout the frequency range. With our optimized apparatus, we could measure the photocurrent components from oriented purple membrane with lifetimes from 70 ms to 32 ns without distortion by the measuring system.     

The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

Application of near-field scanning optical microscopy in photosynthesis research

Many different methods have been developed in recent years to gain insight into the structure of proteins, membranes, organelles and cells. Here we demonstrate the application of near-field scanning optical microscopy (NSOM) for analysis of the structures of typical photosynthetic membrane objects such as chloroplasts and thylakoids from spinach and chromatophores from purple bacteria. To our knowledge, this is the first report of application of NSOM to imaging chromatophores from photosynthetic bacteria and intact thylakoids from higher plants. NSOM has the ability to measure optical signals originating from the sample with a spatial resolution better than conventional optical microscopy. The main advantage of near-field optical microscopy, besides the improved lateral optical resolution, is the simultaneously acquired topography. We have applied NSOM to thylakoids obtained by osmotic shock of chloroplasts. Swollen thylakoids had average diameters of 0.8–1 micron and heights of 0.05–0.07 micron. We also describe the use of fluorescent dyes for the analysis of structures resulting from fusion of photosynthetic bacterial chromatophores with lipid impregnated collodion membranes. The structures formed after fusion of chromatophores to the collodion film have diameters ranging from 0.2 to 10 microns and heights from 0.01 to 1 micron. The dual functionality (optical and topographical), high spatial resolution, and the possibility to work with wet samples and under water, make NSOM a useful method for examining the structures, sizes, and heterogeneity of chromatophore and thylakoid preparations.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Genetic cloning and functional expression in Escherichia coli of an archaerhodopsin gene from Halorubrum xinjiangense

Pairs of PCR primers that targeted the archae/bacteriorhodopsin gene were used to clone the archaerhodopsin (aR) gene of Halorubrum xinjiangense strain BD-1^T, and this gene was sequenced and functionally expressed in Escherichia coli. Recombinant E. coli cells harboring the plasmid carrying this gene became slightly purple or blue depending on whether they were supplemented with all- trans retinal or 3,4-dihydroretinal, respectively, during induction with IPTG. The purple and blue membranes from the recombinant E. coli showed maximal absorption at 555 and 588 nm, respectively, which are different from maximal absorption at 568 nm of the wild-type purple membrane. Purple membranes from the recombinant E. coli and from strain BD-1^T were investigated in parallel. The E. coli purple membrane was fabricated into films and photoelectric responses were observed that depended on the light-on and light-off stimuli.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

缬氨霉素对紫膜碎片双层平板脂膜(BLM)光电响应的影响

本实验中测量了缬氨霉素对双层平板膜系统的电性质和紫膜碎片BLM上光电响应的作用.实验结果表明:缬氨霉素对K~+很灵敏,它可使大豆磷脂BLM的膜电阻下降近四个量级,膜的稳定性也下降.它对Na~+也有作用,但其作用不如K~+那么明显.在缬氨霉素作用下,K~+对紫膜碎片BLM光电响应的影响十分明显,它可以使光电压完全消失.Na~+对紫膜碎片BLM的作用则比K~+的作用小得多.     

Measuring local surface charge densities in electrolyte solutions with a scanning force microscope

To show that local surface charge densities can be measured with a scanning force microscope purple membranes adsorbed to alumina were imaged in electrolyte solutions. Force versus distance curves were measured on purple membranes and on the bare alumina with standard silicon nitride tips. By comparing the electrostatic force measured on both substances, the surface charge density of purple membranes could be calculated from the known charge density of alumina. The charge density of purple membranes was estimated to be -0.05 C/m².     

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. II. Kinetic studies

Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M₄₁₂ evolution. In addition, by monitoring the kinetic resonance Raman spectra in ²H₂O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M₄₁₂. Furthermore, the kinetic spectra of fully deuterated purple membranes in H₂O have allowed us to assign the 1465 cm⁻¹ band in these spectra to the C=C stretching frequency of BR₅₇₀ and the 1512 cm⁻¹ band to the C=C stretching frequency of M₄₁₂. These spectra have also provided an indication of a Raman spectral feature associated with O₆₄₀ and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.     

Photochemically induced charge separation occurring in bacteriorhodopsin. Detection by time-resolved dielectric loss.

Time-resolved dielectric loss (TRDL) measurements are reported for the photochemical excitation of bacteriorhodopsin (bR) in solid films of Halobacterium halobium purple membranes. These measurements provide an independent confirmation for the existence of an important component of charge separation in these membranes after photochemical excitation. The separation of charge is detected by the absorption of microwave energy by the multilayer films of purple membranes in a microwave cavity during flash photolysis experiments. The TRDL method has the advantage of being sensitive to charge separation occurring in both oriented and unoriented films of purple membranes. One disadvantage is that the water content of the samples must be minimized, however, there is some absorbed water present in our electrodeposited solid film samples. To the best of our knowledge, TRDL measurements have not been reported previously for photochemical charge separation in biological membranes. It is significant that an early decay component of TRDL in the 20-microseconds time domain corresponds to the relaxation of the negative charge displacement photocurrent in oriented samples of purple membranes. In addition, a component of charge separation persists during the first several hundred microseconds of the bR photocycle.     

温度对脂质囊泡中单体菌紫质分子结构和光电响应的影响

本实验用人工双分子平板膜系统(BLM)测量了紫膜碎片和在DMPC脂质襄泡膜中的单体菌紫质分子的光电响应以及与温度的关系(处理温度17℃至31℃).温度对紫膜碎片的光电响应影响不大,但对单体菌紫质分子的光电响应有明显影响.用园二色(CD)方法相应地观察了温度对紫膜碎片和单体菌紫质分子在可见波长范围内的CD谱的影响 同样观察到温度对单体菌紫质分子的CD谱有明显影响.两者的影响很可能与脂质襄泡中DMPC的相变温度有关.     

Influence of stray capacitance and sample resistance on the kinetics of fast photovoltages from oriented purple membranes

Flash-induced photovoltages were measured with metal electrodes in two experimental systems of purple membranes oriented by an electric field. One system consisted of a suspension of purple membranes cooled to 80 K. The photovoltage evoked by a xenon flash lamp displayed a single phase with a fast rise and a slow RC-decay. The signal shape is consistent with a fast charge separation occurring before the decay of the K-intermediate. The other system consisted of purple membranes embedded and stabilized in polyacrylamide gel. At room temperature, the photovoltage, evoked by a 10 ns laser flash, displayed a negative phase in the submicrosecond range and a slower positive one. The shape of the signals were altered in a complex manner by the stray capacitance and the ionic strength. The rise-time of the negative phase was approx. 14 and approx. 40 ns at ionic strengths of 10 and 1 mM, respectively. The initial peak amplitudes of the photovoltage from both experimental systems depended on the external capacitance in an inverse manner, indicating that both experimental systems were not impedance-matched. The evaluation of kinetic data of molecular reactions from measurements of the photovoltage is discussed.     

Fourier-transform infrared studies on cation binding to native and modified purple membranes

Fourier-transform infrared spectroscopy has been used to examine the structural differences in the protein moiety between the native purple and the deionized blue membranes, both at pH 5.0. The spectra demonstrate that deionization of purple membrane decreases the content of the distorted alpha II-helices in favor of the more common alpha I-helices. Changes in the signals from beta-turns are also observed. The changes corresponding to the carboxyl groups suggest that deionization leads to a decrease in the strength of the hydrogen bonds involving carboxyl groups. Most of these effects are reversed progressively upon binding of one to five Mn2+ per bacteriorhodopsin to the deionized membrane. Binding of Hg2+ to the deionized membranes does not restore the purple color but induces global changes similar to, but less intense than, those brought about by Mn2+ binding. However, the effects attributed to the carboxyl groups are opposite to those found for Mn2+. Schiff base reduction or bleaching induces a decrease of the content of the alpha II-helix in favor of the alpha I-helix and a decrease in the strength of hydrogen bonds to carboxyl groups. Deionization of these modified membranes leads to a further loss in the alpha II content. These results indicate a conformational rearrangement of the protein structure between the native purple membrane and the deionized membrane, which could arise from surface potential changes elicited by bound cations. The changes observed in the carboxyl groups suggest that some of them are located structurally close to the retinal environment and may be involved in cation binding.     

Structure and hydration of purple membranes in different conditions

The unit cell dimension of the bacteriorhodopsin lattice in purple membranes decreases by the same amount (2%) upon drying the membranes at room temperature as when they are cooled to liquid nitrogen temperatures. Neutron diffraction experiments with H2O:2H2O exchange, however, show that whereas in the dry membranes the lipid headgroups are dehydrated and the decrease in dimension is due to a smaller area occupied by the lipid molecules, the water of hydration remains in place in the cooled membranes, and the decrease in dimension is due to thermal contraction only. These data suggest a hypothesis that functional bacteriorhodopsin, in the wet state at room temperature, has a relatively soft environment that would allow large amplitude motions of the protein; in the dry membranes at room temperature (which are inactive), the amplitudes of protein motions would be inhibited by a more close-packed environment as they are reduced, due to thermal contraction, in the cold membranes.     

GTP hydrolysis by tubulin occurs with water oxygen incorporation into inorganic phosphate

Tubulin assembly was conducted in [18O]H2O and the resulting mixture of GTP, GDP and Pi was examined by 31P-NMR. Two Pi signals, separated by about 0.02 ppm, were observed. By combining this mixture with a solution of Pi containing all five possible 16O and 18O isotopomers of Pi, it was shown that the two signals were due to [16O4]- and [16O3, 18O]Pi. The amount of 18O incorporated into the Pi was that expected if the hydrolysis of GTP during tubulin assembly occurs with cleavage of the gamma-phosphorus-bridge oxygen bond.     

Characteristics and mechanisms of the two types of photoelectric differential response of bacteriorhodopsin-based photocell

Bacteriorhodopsin (BR)-based photocells have been assigned possessing differential photoelectric response. But recently we found that the differential response described before, which occurred in milliseconds to seconds, outputting a positive pulse when light was on and a negative pulse when light was off, was not the intrinsic property of the BR molecule. It was partially caused by the measuring circuit. By measuring the photoelectric response signal of the BR film photocell to a short laser pulse, the impulse response function of the BR film photocell was obtained by data fitting with MATLAB software. A simulation system was accordingly developed. The output response signals of the BR film photocell under different stepping incident light were calculated. By simulation and analysis, it was concluded that the differential response caused by the intrinsic property of the BR molecule happened in microseconds time scale, and it produced a negative pulse when light was on and a positive pulse when light was off. It was much faster but much weaker than that described before.     

Time-resolved resonance Raman characterization of the bO640 intermediate of bacteriorhodopsin. Reprotonation of the Schiff base.

The resonance Raman spectrum of photolyzed bacteriorhodopsin under conditions known to increase the concentration of the bO640 intermediate in both H2O and D2O is presented. By use of computer subtraction techniques and a knowledge of the Raman spectra of the unphotolyzed bacteriorhodopsin as well as the other intermediates in the cycle, a qualitative spectrum of bO640 is determined. The shift of a band at 1630 cm-1 in H2O to 1616 cm-1 in D2O suggests that the Schiff base of bO640 is protonated. Additional bands at 947, 965, and 992 cm-1 that appear only in D2O suspensions confirm that a proton is coupled to the retinal chromophore of bO640. The reprotonation of the Schiff base thus occurs during the bM412 to bO640 step. The fingerprint region, sensitive to the isomeric configuration of the retinal chromophore of bO640, is dissimilar to the fingerprint regions of published model compounds and other forms of bacteriorhodopsin.