Krzysztof Bryl

Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes.

This paper presents the application of chronopotentiometry in the study of membrane electroporation. Chronopotentiometry with a programmable current intensity was used. The experiments were performed on planar bilayer phosphatidylcholine and cholesterol membranes formed by the Mueller-Rudin method. It was demonstrated that a constant-intensity current flow through the bilayer membranes generated voltage fluctuations during electroporation. These fluctuations (following an increase and decrease in membrane conductance) were interpreted as a result of the opening and closing of pores in membrane structures. The decrease in membrane potential to zero did not cause the pore to close immediately. The pore was maintained for about 200 s. The closing of the pore and recovery of the continuous structure of the membrane proceeded not only when the membrane potential equalled zero, but also at membrane potentials up to several tens of millivolts. The fluctuations of the pore were possible at values of membrane potential in the order of at least 100 mV. The size of the pore changed slightly and it closed after some time below this potential value.

Chronopotentiometric studies of electroporation of bilayer lipid membranes.

The constant-intensity current chronopotentiometric measurements of egg yolk phosphatidylcholine bilayer membranes (BLM) are presented. It is demonstrated that a constant-intensity current flowing through the bilayer membranes generates the pores in their structures. For the current intensity from 0.1 to 2.0 nA, the generated pores open and close cyclically. The frequency of oscillations depends on the current intensity: the higher current intensity, the higher frequency of pore oscillations. It is suggested that the presented method may allow to create one pore in BLM and to observe its dynamical behaviour. Based on chronopotentiometric curves, a method of pore conductance calculations is presented. It is demonstrated that the value of obtained conductance can be applied for pore diameter estimation. The hypothetical application of constant-current method as a biotechnological tool for selective and controlled incorporation of molecules into microorganisms is discussed.

FEMTOSECOND STUDIES OF PRIMARY PHOTOPROCESSES IN OCTOPUS RHODOPSIN

Abstract— Femtosecond spectroscopy of octopus rhodopsin in H2O and D2O was performed over a very wide spectral region of 400–1000 nm. Transient gain and absorption from the excited state were observed for the first time around 650 and 700 nm, respectively, just after 300 fs pulse excitation. Bathorhodopsin was formed within 400 fs from the excited state; therefore, the cis‐trans isomerization completes within 400 fs. The first intermediate “primerhodopsin” found in our previous paper is most likely “quasi‐thermal” bathorhodopsin, in which the local thermalization of the chromophore is achieved. Then cooling down of the chromophore to the surrounding protein temperature takes place with 20 ± 10 ps along with blue‐shifting of a spectrum of 10 ± 5 nm. In addition to these observations, a prominent gain in the region of > 850 nm was observed and decayed with 2–3 ps in H2O. A similar time constant was estimated for a partial decay of an induced absorption around 600 nm. This process may be related with two forms of bathorhodopsin reported previously. In this scheme, two forms of bathorhodopsin are formed with time constants of about 400 fs and 2 ps. In the sample in D2O, time constant of 3–4 ps was obtained for the slower process.

Changes of structural and dynamic properties of model lipid membranes induced by α-tocopherol: implication to the membrane stabilization under external electric field

The effects of alpha-tocopherol on electric properties of bilayer lipid membranes were investigated. Planar bilayer membranes formed by the Mueller-Rudin method were used. Voltammetric and chronopotentiometric measurements were performed using a four-electrode potentiostat-galvanostat. It was demonstrated that registration of membrane capacitance, resistance, and voltammetric characteristics provided information about the change in the structure and permeability of bilayer lipid membranes. The results suggested that incorporation of alpha-tocopherol into lipid membrane destabilized its structure and facilitated the electrogeneration of pores. The possible role of observed changes in physiological functions of alpha-tocopherol was discussed.

The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA—H2O mixture. The bacteriorhodopsin photocycle in the PVA—H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412‐O640 and O640‐bRa11‐trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.

Light- and dark-adaptation of bacteriorhodopsin measured by a photoelectric method

Abstract The process of light- and dark-adaptation of bacteriorhodopsin was studied by measuring the photoelectric response signal corresponding to the M form of the light-adapted photocycle. Two different kinds of sample were used: oriented dried samples and oriented gel samples. It was shown that the quantum efficiency of light-adaptation does not depend on the temperature of the sample, but strongly depends upon the water content, which means that in this process the determining step of the branching is the light excitation (bacteriorhodopsin to bacteriorhodopsin ∗ or bacteriorhodopsin ∗ to the next state), not a later step during the 13-cis photocycle. The process of dark-adaptation depends upon both the temperature and the humidity of the sample.

The role of retinal in the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles

Abstract. The effects of bacteriorhodopsin analogues and the analogues of a bacteriorhodopsin mutant (D96N) on the lateral organization of lipids have been investigated with lipid species with a variety of acyl chain lengths. The analogues, obtained by regeneration of bacterioopsin or mutant opsin with 14-, 12-, 10-, or 8-fluororetinal, were reconstituted with 1,2-didodecanoyl-sn-glycero-3-phosphocholine, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine. The phase behavior of the protein-lipid systems was investigated at different temperatures and different protein/lipid molar ratios by analyzing the fluorescence and phase properties of the 1-acyl-2-[8-(2-anthroyl)octanol]-sn-glycero-3-phosphocholine probe. The (8,10,12)-bacteriorhodopsins had a similar effect on the lipid phase transition to that induced by native bacteriorhodopsin: a rigidifying effect on the three shorter lipid species and a fluidifying effect on the longest-chain lipids used. The substitution of retinal with 14-fluororetinal resulted in much stronger effects of the protein on the lipids: a more pronounced up-shift of the lipid phase transition temperature, a rigidifying effect on all the lipids used, and an elongation of the distance over which the hydrophobic thickness of the lipid bilayer was perturbed by the protein. Evidence was provided that retinal contributed to the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles. The extent of this contribution was dependent on the retinal structure in close vicinity to the Shiff base and on the compactness of the protein structure.

Retinyl palmitate in water environment

Abstract The interaction between retinyl palmitate molecules and their environment in binary water- p -dioxane solvent (at different molar ratios of water to p -dioxane) was studied. The absorption, fluorescence emission and fluorescence excitation spectra were analysed. In this binary solvent, the interaction between retinyl palmitate molecules and environments of different polarity leads to the self-organization of retinyl palmitate. The possible consequences of the existence of self-organized structures for retinoid transport through the water phase are discussed.

A Water Environment Forces Retinyl Palmitate to Create Self-Organized Structures in Binary Solvents

The interaction between retinyl palmitate molecules and their environment in binary water/p-dioxane and water/isopropyl alcohol solvents was studied under different solvent polarities. The binary solvent polarities were changed by changing the molar ratios of water to p-dioxane (or isopropyl alcohol). The fluorescence emission and fluorescence excitation spectra were analyzed. In binary solvents, the interaction between retinyl palmitate molecules and environments of different polarities leads to the self-organization of retinyl palmitate, and as a result, different fluorescence centers are created. The similar fluorescence properties of these centers in different binary solvents were interpreted as a water driving force inducing the self-organization of retinyl palmitate. The possible consequences of this phenomenon for misleading interpretation of model studies of retinol interaction with retinol transporting proteins are also discussed.

Mechanism of the Leakage Induced on Lipid Model Membranes by Rz1 Lipoprotein, the Bacteriophage λ Rz1 Gene Product: A Fluorescence Study

The leakage of aqueous contents of neutral (dipalmitoylphosphatidylcholine/cholesterol) liposomes as induced by Rz1, a proline-rich lipoprotein, the bacteriophage λ Rz1 gene product, was studied. Fluorescence enhancement assay with Tb3+/dipicolinic acid and self-quenching assays with carboxyfluorescein and fluorescein isothiocyanaten-dextran were used to monitor the Rz1-induced leakage from neutral liposomes. The results demonstrated that Rz1 caused local membrane destabilization leading to the leakage of the liposome contents. The extent of the leakage was independent of the molecular mass of the liposome-entrapped solutes in the range of 376–4000 Da. The kinetics of Rz1-liposome leakage was very similar to that obtained with detergent Triton X-100 for all the solutes used. The results suggested that Rz1 can act as a detergent; i.e., by interacting with lipids on both sides of the liposome membranes (inducing perturbation in the lipid packing within the bilayer), it facilitates the transfer of encapsulated molecules into the external liposome environment. The importance of this result for Rz1 physiological function is discussed.