Location of two sulfhydryl groups in the rhodopsin molecule by use of the spin label technique

Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin. These groups are located in protected but probably different environments, less then 12 Å away from the aqueous phase. A distance of about 37 Å is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phospholipid-protein interactions.     

Fluid lipid fraction in rod outer segment membrane

Rod outer segment membrane is analyzed using the spin label technique by means of two probes. The solubility of the first label, 2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Fluid lipid fraction in rod outer segment membrane.

Rod outer segment membrane is analyzed using the spin resonance label technique by means of two probes. The solubility of the first label,2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Photoacoustic spectroscopy of bacteriorhodopsin photocycle

Photoacoustic spectroscopy was applied to study the energetics and the kinetics of the slow intermediates of the bacteriorhodopsin photocycle. An analysis of the modulation frequency dependence of the photoacoustic signal allowed us to estimate the enthalpy changes and the kinetic parameters associated with those intermediates. The effects of pH, salt concentration, and protein aggregation were studied. Three photoacoustic transitions were found. The two low frequency transitions were attributed to O660 and M412, respectively. The third transition was interpreted as resulting from a protein conformational change undetected spectrophotometrically. The frequency spectra were simulated between 5 and 180 Hz at pH's 5.1, 7.0, and 8.9 assuming a branching in the bacteriorhodopsin photocycle at the M412 level. The enthalpy changes associated with M412 and O660 were computed and compared with the experimental values.     

Quantum efficiency of light-driven proton extrusion in Halobacterium halobium. pH dependence.

The quantum yield for light-induced proton extrusion in Halobacterium halobium cells pretreated with an ATPase inhibitor was measured between pH 5 and 9 using two separate spectrophotometric techniques. The transmittance of the cell suspension was measured with a spectrometer equipped with "end-on" photomultipliers, whereas the reflectance was measured using a light-integrating sphere. The potentialities of the two techniques are critically compared. These measurements are used to evaluate the intensities of light absorbed by the cells. Since the initial rates of proton release into the extracellular medium were simultaneously measured, the quantum yield values [QY(H+)] could be determined. The results obtained with the two techniques are in reasonable agreement. QY(H+) is 0.64 at pH 5.9 and decreases gradually to 0.28 at alkaline pH values.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Electrochemical Performance Enhancement of 3D Printed Electrodes Tailored for Enhanced Gas Evacuation during Alkaline Water Electrolysis

A zero-gap cell with porous electrodes is a promising configuration for alkaline water electrolysis. However, gas evacuation becomes a challenge in that case, as bubbles can get trapped within the electrode's 3D structure. This work considers a number of 3D printed electrode geometries with so-called triply periodic minimal surfaces (TPMS). The latter is a mathematically defined structure that repeats itself in three dimensions with zero mean curvature, and can therefore be expected to be particularly well-suited to enhance gas evacuation. Another advantage as compared to other state-of-the-art 3D electrodes like foams or felts lies in the fact that their porosity, which determines the available surface area, and their pore size or flow channel dimensions, which determines the degree of bubble entrapment, can be varied independently. By a combined experimental and modeling approach, this work then identifies the structural parameters that direct the performance of such 3D printed TPMS geometries toward enhanced gas evacuation. It is demonstrated that an optimal combination of these parameters allows, under a forced electrolyte flow, for a reduction in cell overpotential of more than 20%. This indicates that efforts in optimizing the electrode's geometry can give a similar electrochemical performance enhancement as optimizing its electro-catalytic composition.