Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Is the purple color of bacteriorhodopsin maintained by lipid-protein interactions?

When bacteriorhodopsin is delipidated and purified in detergents, its purple chromophore can be reversibly titrated to a red one. The pK_a of this equilibrium depends on the nature of the detergent in which bacteriorhodopsin is dispersed. In the absence of solvating amphiphiles, lipid-free detergent-free bacteriorhodopsin is red (λ_max = 480 nm) at pH higher than 3.5.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

Temperature and pH sensitivity of the O640 intermediate of the bacteriorhodopsin photocycle

The temperature and pH dependencies of the O₆₄₀ intermediate of the photocycle of bacteriorhodopsin (bR) were investigated by flash photolysis and T-jump experiments. The maximal concentration of the O₆₄₀ intermediate was found to be dependent on the temperature, which is described by a sigmoidal relationship. With increasing pH the midpoint of the sigmoidal curves shifts to higher temperatures. The Van't Hoff equation provides enthalpy and entropy values of the observed states. These results indicate that, in the investigated temperature (0-60°C) and pH (pH 4.0-10.0) range, the sequence of the principal intermediates in the pathway “M-N-O-bR” does not change. The observations of the O₆₄₀ intermediate at pH < 8.0 and of the N₅₅₀ intermediate at pH > 8.0 are most probably due only to changes of the intrinsic rate constants of the bR photocycle, not to a different mechanism.     

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Number and size distribution of the DNA chains in Escherichia coli.

The essential replication protein encoded by gene O of bacteriophage λ (O-λ) is one of the major polypeptides produced in vitro in a DNA-dependent protein synthesizing system with λ DNA as template (Yates et al., 1977). We have used this system to identify the proteins encoded by lambdoid phages φ80 and 82 and equivalent in function to O-λ. The O protein of each phage type differs slightly in polypeptide molecular weight. Hybrid λ-φ80 and λ-82 phages derived by recombination within gene O direct synthesis of hybrid O proteins with the aminoterminal segment characteristic of one parent, and the carboxyl-terminal segment characteristic of the other. Differences in structure among O-λ, O-80 and O-λ82 segregate together with specificity determinants for interactions between the O protein and the control site ori, and between the O protein and the product of replication gene P. The coding region for the O protein includes ori.     

Probing membrane protein unfolding with pulse proteolysis

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.     

Effects of UV-Radiation on Kinetics of Encystment and on Morphology of Resting Cysts of the Ciliate Laurentiella acuminata1

The effects of ultraviolet radiation (λ= 254 nm) on the kinetics of encystment of the hypotrichous ciliate Laurentiella acuminata and the structure of resting cysts obtained from irradiated precystic cells are reported. High doses of UV-radiation caused a delay of encystment with a linear increase in the average time for obtaining 50% of encystment (EN₅₀). Resting cysts with abnormal cyst walls were obtained when precystic cells were irradiated in the exposure range 720 to 960 J/m². The cystic layer (mesocyst) was approximately twice as thick (6.5 μ m) as normal (3.7 μ m). Microscopical observations of abnormal cysts revealed the presence of two complete mesocysts, and the absence of the spines characteristic of the ectocyst. The UV-dependent effects on the cyst wall were gradually corrected in successive generations of the irradiated cells.     

EFFECTS OF TEMPERATURE ON GROWTH,LIGHT ABSORPTION,AND QUANTUM YIELD IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of growth temperature on the marine chlorophyte Dunaliella tertiolecta Butcher were studied to provide a more mechanistic understanding of the role of environmental factors in regulating bio-optical properties of phytoplankton. Specific attention was focused on quantities that are relevant for modeling of growth and photosynthesis. Characteristics including chlorophyll a (chl z)-specific light absorption (a*_ph(λ)), C:chl a ratio, and quantum yield for growth (φ_μ) varied as functions of temperature under conditions of excess light and nutrients. As temperature increased over the range examined (12°-28°C), intracellular concentrations of chl a increased by a factor of 2 and a*_ph(λ) values decreased by more than 50% at blue to green wavelengths. The lower values of a*_ph(λ) were due to both a decrease in the abundance of accessory pigments relative to chl a and an increase in pigment package effects arising from higher intracellular pigment concentrations. Intracellular pigment concentration increased as a consequence of higher cellular pigment quotas combined with lower cell volume. At high growth temperatures, slightly more light was absorbed on a per-cell-C basis, but the dramatic increases in growth rate from μ= 0.5 d^?1 at 12° C to μ= 2.2 d^?1 at 28°C were primarily due to an increase in φ_μ (0.015–0.041 mol C (mol quanta)^?1). By comparison with previous work on this species, we conclude the effects of temperature on a*_ph(λ) and φ_μ are comparable to those observed for light and nutrient limitation. Patterns of variability in a*_ph(λ)and φ_μ as a function of growth rate at different temperatures are similar to those previously documented for this species grown at the same irradiance but under a range of nitrogen-limited conditions. These results are discussed in the context of implications for bio-optical modeling of aquatic primary production by phytoplankton.     

Distributed Kinetics of the Charge Movements in Bacteriorhodopsin: Evidence for Conformational Substates

The flash-induced charge movements during the photocycle of light-adapted bacteriorhodopsin in purple membranes attached to a black lipid membrane were investigated under voltage clamp and current clamp conditions. Signal registration ranged from 200 ns to 30 s after flash excitation using a logarithmic clock, allowing the equally weighted measurement of the electrical phenomena over eight decades of time. The active pumping signals were separated from the passive system discharge on the basis of an equivalent circuit analysis. Both measuring methods were shown to yield equivalent results, but the charge translocation could be accurately monitored over the whole time range only under current clamp conditions. To describe the time course of the photovoltage signals a model based on distributed kinetics was found to be more appropriate than discrete first order processes suggesting the existence of conformational substates with distributed activation energies. The time course of the active charge displacement is characterised by a continuous relaxation time spectrum with three broad peaks plus an unresolved fast transient (<0.3 μs) of opposite polarity. The time constants and relative amplitudes (in brackets) derived from the peak rate constants and relative areas of the three bands are: τ₁ = 32 μs (20%), τ₂ = 0.89 ms (15%) and τ₃ = 18 ms (65%) at 25°C in 150 mM KCl at pH7. The Arrhenius plots of the peak rate constants were linear yielding activation energies of E_A1 = 57 kJ/mol, E_A2 = 52 kJ/mol, and E_A3 = 44 kJ/mol. The electrical signal at 890 μs has no counterpart in the photocycle of bacteriorhodopsin suspensions. Fits with a sum of exponentials required 5 to 6 components and were not reproducible. Analysis of photoelectrical signals with continuous relaxation time spectra gave equally good fits with fewer parameters and were well reproducible.     

Equilibrium and kinetics of thermal unfolding of yeast 5.8S ribosomal RNA in aqueous salt solutions

Equilibrium and kinetics of thermal melting of yeast 5.8S ribosomal RNA in aqueous NaCl were investigated by differential thermal melting and temperature jump methods. Two peaks were observed in each of the melting curves at 1 mM-1 M Na⁺ and linearity between each melting temperature T_m and log[Na⁺] was found at [Na⁺> 10 mM. From the difference spectrum ratio, $\text{d}\text{A}{}_{280}\text{d}\text{A}{}_{260}$, the G-C content in the local structures was calculated to be 91 and 56%. The temperature jump to 70–85°C in aqueous 30 mM Na⁺ of the RNA solution induced first-order kinetics, from which the kinetically determined melting curve was calculated. The curve could be approximately described in a Gaussian form with a T_m which agrees well with the high T_m in the static melting curve at 30 mM Na⁺. The kinetic properties of the reaction indicated a double helix-coil transition. However, the temperature jump to 20–60°C did not induce monophasic kinetics. The kinetic amplitude of the slow component showed a T_m which corresponded to the low T_m in the static melting curve at 30 mM Na⁺. The slow relaxation had the characteristics of a double helix-to-coil transition. However, contributions from very fast processes including single strand unstacking, were most noticeable in the low temperature melting region of the static curve. The thermodynamic parameters of both transitions from double helix to coil were analysed in detail. Both activation energies for helix formation were negative, and the nucleation is thought to follow a process similar to that in oligonucleotides. Values of T_m and enthalpy change of both helix-coil transitions indicated the cloverleaf model as the most plausible one for some limited regions of yeast 5.8S RNA among the previously proposed models: burp gun, cloverleaf and Rubin's models.     

Structure and siderophore activity of ferric schizokinen

Schizokinen, a citrate-containing dihydroxamate, is a siderophore produced by Bacillus megaterium and Anabaena sp. The involvement of the citrate α-hydroxycarboxylate moiety in iron chelation was investigated by comparing the iron binding behavior of schizokinen with that of acetylschizokinen, a derivative in which the citrate hydroxyl group was modified by acetylation. Ferric schizokinen was found to exhibit an absorption spectrum (λ_max = 460 nm) characteristic of a dihydroxamate below pH 2.5, with an isosbestic shift to a citrate dihydroxamate spectrum (λ_max = 395 nm) above pH 4. Ferric acetylschizokinen also had a dihydroxamate absorption spectrum (λ_max = 465 nm) at low pH. However, its spectral shift (λ_max = 420 nm) and intensity above pH 4 were more typical of a ferric trihydroxamate. The molecular weight and electrophoretic mobility of ferric acetylschizokinen are consistent with a dimeric Fe₂ (acetylschizokinen)₃ structure, whereas ferric schizokinen appears to exist as a monomeric 1:1 complex Despite the differences in molecular weight and α-hydroxycarboxylate coordination, both complexes are effective in promoting iron uptake in Anabaena.     

Oxidative dehydrogenation of an iron-tetraazacyclotetradecatriene complex

The oxidative dehydrogenation of the bis(N- methyl imidazole)(meso-5,5,7,12,12,14-hexamethyl- 1 4,8 11-tetraazacyclotetradeca-1,3,8-triene)iron(II) complex, forming the corresponding 1 3,8,10-tetraene product was investigated by cyclic voltammetry, spectroelectrochemistry and stopped-flow kinetics with [Fe(CN)₆]³⁻, at 25 °C, I = 0.50 M and pH 7–10. The results led to a mechanism consistent with a reversible one electron transfer process generating iron(III) species which lose a proton (pK_a = 9.77) and undergo induced electron transfer in the presence of the hexacyanoferrate(III) ion (k = 4.2 × 10⁵ M⁻¹ s⁻¹). The intermediate precursor complex (λ_max = 665 nm) formed at this step, converted to the tetraene product according to a first order kinetics, with k = 0.12 s⁻¹.     

Different rotamer populations around the C-5C-6 bond for α- and β-d-galactopyranosides through the combined interaction of the gauche and anomeric effects: A 300-MHz 1H-n.m.r. and mndo study

A 300-MHz ¹H-n.m.r. study of methyl 2,3,4-tri-O-methyl-ga- (1) and β-d-galactopyranoside-6-(dimethyl phosphate) (3), using various solvents, shows that the gauche (gg) rotamer populations about the C-5C-6 bond are are the same in all solvents, whereas those of the gauche(trans) (gt) and trans(gauche) (tg, O-5 and O-6 trans) rotamers are solvent dependent. The tg population increases with decreasing polarity of the solvent, which is attributed to an increased electrostatic repulsion between O-5 and O-6 in apolar solvents. The tg population of 3 is larger than that of 1 and the same difference is observed in the corresponding compounds (2 and 4) which have a trigonal-bipyramidal five-coördinated phosphorus (P^v) at position 6 and which have a higher electron density at O-6. These differences in rotamer populations are due to an effect additional to that of the coulombic effect between O-5 and O-6. That these differences are caused by a combination of the gauche and anomeric effects is supported by the finding that the tg population increases with increasing pK_a of the group at C-1. The results of the n.m.r. measurements (in CCl₄) are reproduced fairly accurately by MNDO calculations on model systems. The solvent dependence of the rotamer population around the C-5ẋC-6 bond is a good criterion for the assignment of the H-6S,6R resonances since, for galactopyranosides, J_5,6S increases and J_5,6R decreases as the polarity of the solvent decreases.     

Ribosome changes during translation

Studies on the quantitative binding of [³H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [³H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by K_d = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by K_d,H = 0.81 μm and K_d,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K_d^CHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of K_d^DC = 0.82 μm. These dissociation constants are identical to K_d,L and K_d,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by K_d^DT = 1.5 μm and K_d^FA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.     

Temporal Modulation of Sodium Current Kinetics in Neuron Cells by Near-Infrared Laser

Near-infrared laser provides a novel nerve stimulation modality to regulate the cell functions. Understanding its physiological effect is a prerequisite for clinic laser therapy applications. Here, the whole-cell sodium (Na) channel kinetics of neuron cell was employed to determine the temporal roles of infrared laser. The Na currents were elicited by electrical pulses that were synchronized at the rising and falling edges of the 980 nm laser pulses, respectively, to investigate the different infrared effect on cell functions. The time constants of activation (τ _m) and inactivation (τ _h) kinetics were extracted from fitting of the Na current (m³h) according to the Hodgkin–Huxley (HH) model. By comparing the time constants without and with the laser irradiation, we obtained that laser pulses changed the Na current kinetics by accelerating τ _h-phase and slowing down τ _m-phase at the beginning of the laser pulse, whereas both phases were accelerated at the end of the pulse. After relating the ratios of the time constants to the temperature characteristics of Na channel by Q ₁₀, we found that the accelerating in Na current kinetics could be related to the average temperature of extracellular solution in the corresponding time span by choosing Q ₁₀ = 2.6. The results of this study demonstrated that there was a positive correlation between the acceleration of the Na current kinetics and increases in temperature of the extracellular solution.     

The utility and performance of near-infra red spectroscopy in simultaneous monitoring of multiple components in a high cell density recombinant Escherichiacoli production process

Bioprocess optimisation is often limited by an inability to measure biomass, nutrient and by-product concentrations in a time frame which allows process adjustments. Near-infrared (near-IR) spectroscopy can potentially be used to measure each of these components within 2 minutes of sampling, using an unprocessed whole broth sample. In the present study the use of near-IR spectroscopy for at-line (rapid off-line) monitoring of biomass, glycerol, ammonium, and acetate in a recombinant Escherichia coli fed batch process was investigated. The following robust correlation models were developed for these analytes using multiple least squares linear regression (MLR): [Glycerol],gl^?1 =15.957 ? 2219.270^* A(λ₂₂₇₄)?1705.041^* A(λ₂₁₇₂); [Acetate],gl^?1=27.683 ?1757.258^* A(λ₂₂₅₄)+296.903^* A(λ₂₃₄₀)?21.325^* A (λ₆₂₀); [Ammonium],gl^?1=?1310.502?47912.960^* A (λ₂₁₄₈)?135149.300^* A(λ₁₇₈₂)?27636.200^* A (λ₈₃₀); and [Biomass],gl^?1=14.034?3.548^* A(λ₆₀₂/λ₁₁₃₄) ?4286.050^* A(λ₉₂₈). Using these models permitted rapid simultaneous analysis of all four analytes. This improved monitoring capability was used to develop a high cell density recombinant E. coli fed-batch process in which ammonium and acetate accumulation were minimised leading to higher cell densities. By manipulation of the C?:?N ratio in the complex feed, the toxic effects of ammonium accumulation upon the organism were minimised, thereby facilitating the application of a carbon limited feeding strategy. The effect of these C?:?N ratio medium changes, upon the near-IR measurement capability, was investigated. In this process, near-IR spectroscopy has been shown to be a powerful, accurate and precise method for simultaneously measuring several key process variables. Its accuracy, precision and utility for at-line measurement and control are evaluated, particularly in reference to processes where the initial medium composition may vary, leading to changes in the chemical matrix. The potential of near-infra red for online analysis and control is discussed.