Béla Karvaly

Incorporation of bacteriorhodopsin into a bilayer lipid membrane; a photoelectric-spectroscopic study

Much research has been done in the past five years on purple membrane fragments (PM) containing a rhodopsin-like protein-pigment complex bacteriorhodopsin (BR) from Halobacterium halobium. In particular biochemical properties of PM have been intensively examined [l-3] . It now seems evident that BR functions as a high-efficiency photoelectric energy transducer, converting light energy to electric and chemical energies; it works as a light-driven proton pump and directs a new type of photophosphorylation [4-71. However, such basic problems as the role of the organized arrangement of BR molecules in membrane-electric phenomena, the origin of the protons and the mechanism of proton translocation under illumination have persistently eluded all attempts at elucidation. Attempts to make model photoreceptor membranes are known in the literature [8-l 11. The disadvantages of these reconstituted membranes were their extremely short lifetime, or they were stable only under nonphysiological conditions when the bilayer structure might have been drastically modified. As first Skulachev’s group was able to incorporate BR into thick (not bimolecular) lipid membranes using proteoliposomes [ 12,131. Recently Shieh and Packer have reported similar experiments on lipid membranes stabilized by polystyrene [ 141. We describe here a new method for the reconstruction of cell membrane structure and function in bimolecular lipid membranes (BLM) with functionally active PM. These BLMs with PM were very stable and exhibited both large-amplitude photovoltaic and photo-

Bacteriorhodopsin: A molecular photoelectric regulator Quenching of photovoltaic effect of bimolecular lipid membranes containing bacteriorhodopsin by blue light

Most studies on the function of bacteriorhodopsin (BR), the purple protein-pigment complex forming the molecular basis of light-energy conversion for a new type of photophosphorylation, have been performed on purified membrane fragments, employing biochemical and photochemical methods [l-8] . BR works as a light-driven vectorial proton-pump mediated by its photochemical cycle: a proton is ejected by the illuminated BR into the extracellular medium while a proton is taken up, during the dark reformation of BR, from the intracellular space [2,9 ] . The reconversion of the bleached BR into purplecomplex can be accelerated by 412 nm illumination, from which the acceleration of turnover number of the proton-pump was postulated [ 1 ,lO-131. That BR behaves as a molecular photoelectric generator was also demonstrated by using proteoliposomes and thin lipid membranes [14-l 61. The reconstruction of certain functions of the Halobacterium halobium envelope in bimolecular lipid membranes (BLM) containing BR in an oriented fashion has quite recently been achieved by the present authors [17--l 91. Although the action spectrum followed the absorption spectrum of BR, the steady-state photoelectric response observed could not be correlated to any elementary step in the photochemical cycle, but rather reflected the overall process. It was noticed that the photovoltaic response of a BR-BLM under whitelight illumination was less than expected on the basis of monochromatic illumination with the 500 nm and 554 nm components of the same white light, respectively [ 17,193 . The present report is concerned with this unusual phenomenon. It is shown that the bleached BR (412 form, which we suggest to call leuco-BR = LBR) under blue-light excitation operates as a proton-sink (or partly as a light-driven reverse proton pump?) for protons produced and pumped through the membrane by the photo-excited BR. This effect is considered to cause the quenching of the photopotential generated by BR irradiated in its green absorption band and thereby, the reduced photoelectric efficiency of white-light. The phenomenon seems to be of fundamental importance in the cellular regulatory mechanism of Halobacteria.

Laser raman studies of molecular interactions with phosphatidylcholine multilayers. II. Effects of mono- and divalent ions on bilayer structure

Laser Raman spectroscopy was applied to characterize structural behavior of dipalmitoyl phosphatidylcholine multibilayer systems in the presence of several cations (K+, Na+, Cs+, Rb+, Ca2+, Mg2+, Cd2+, Ba2+) and anions Cl-, Br-, I-, NO-3, SO2-3, SO2-4, S2O2-3, S2O2-8). To evaluate the Raman-spectroscopical data quantitatively, characteristic intensity ratios, lateral and trans order parameters were used and compared. It was shown that the different trans order parameters are rather sensitive to ion-polar head group interactions and thus, they cannot give unequivocal information on the trans-gauche isomerization of hydrocarbon chains of phospholipids. The observed effects of ions on Raman spectra of phospholipid multilayers could not be explained simply on the basis of electrostatic interactions. The possible involvement of other factors (changes in polarizability, hydrogen bonds, etc.) is also discussed. It was demonstrated that the order parameters defined in different ways may result in different effectiveness sequences of ions. Of monopositive ions Na+ was found to be the most effective to influence the bilayer structure. For dipositive ions, of which Ca2+ proved to be the most effective, concentration-dependent effectiveness sequences were obtained. A plausible interpretation and some consequences of the concentration-dependent two-step binding of divalent cations were also outlined. Bilayered phospholipid structures turned out to be more responsive to anions than to most cations investigated. Interdependent actions of cations and anions, as well as the possible relevance of the charge distribution on anions are postulated.

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. The quenching effect of blue light

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.

Comments on the quantitative interpretation of biomembrane structure by Raman spectroscopy

The possibility of quantitation of information obtained from laser Raman spectra of aqueous lipid dispersions is discussed. It is shown that the all-trans chain order parameter ST introduced by Gaber and Peticolas ((1977) Biochim. Biophys. Acta 465, 260) for the characterization of biomembrane structure is of restricted applicability. This order parameter may give adequate information if polar head groups are not affected at all by the interaction resulting in trans-gauche isomerization. To demonstrate this, data on the effects of mono- and divalent ions on the all-trans chain order parameter are given. The lateral order parameter proved to be suitable for quantitative studies even in the case of ion-head group interaction.

Electronic conduction in bimolecular lipid membranes: exciton mechanism of iodine effect

Abstract The effects of iodine, iodide ion and triiodide ion on the electrical conduction of artificial bimolecular lipid membranes are discussed. It is shown that exciton-exciton and exiton-ion interactions may be responsible for the electronic charge carrier generation at the interface. The dramatic resistance drop of bimolecular lipid membranes in the presence of iodine-iodide is attributed to these exciton processes. A detailed description of the elementary steps of the overall reactions postulated earlier is given. Some aspects of the possible role of charge transfer complexes and of electronic properties of membranes in catalytic processes are outlined.

Lipid-water interaction : exciton states in biological systems?

Abstract The interaction between oxidized cholesterol and water has been investigated by the optical as well as the dielectric method. It was concluded that charge-transfer exciton states may account for the spectral changes in both optical and dielectric spectra and that a hopping-like conduction mechanism operates in both wet bulk lipid samples and bimolecular lipid membranes. The existence of charge-transfer excitons at biological interfaces has been postulated.

Excitons and electronic phenomena at biological interfaces : a new approach to bioelectronic processes

Abstract This paper is primarily concerned with the question of the effect of lipid/water interactions on the electric conductances of lipids and unmodified bimolecular lipid membranes. The problem has been approached by using a phenomenological description in which the existence of charge-transfer excitons was assumed. It is shown that charge-transfer excitons can be held responsible for the resistance-lowering effect of polar adsorbates such as water, etc., and they offer a new possibility to explain the origin of the compensation law observed on either bulk lipid samples or on bilayer lipid membranes. Some possible biological consequences of this special interaction are also discussed.