Tryptophan photolysis is responsible for gramicidin-channel inactivation by ultraviolet light

doi:10.1016/0005-2736(86)90403-7

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 861, 1986, Pages 62-66

https://doi.org/10.1016/0005-2736(86)90403-7 Get rights and content

Abstract

The decay of gramicidin fluorescence resulting from ultraviolet exposure was compared to the decay of conductance from gramicidin-containing planar bilayer membranes under the same conditions of illumination. The decay rate was the same for both processes. The fluorescence decay was identical whether gramicidin was dissolved in methanol or incorporated into lipid vesicles, indicating that the peptide conformation does not affect the sensitivity of gramicidin to photolysis. The correlation of fluorescence decay and conductance decay imply that conductance loss from gramicidin-doped membranes illuminated with ultraviolet light is due to photochemical modifications of the channel tryptophans rather than simply to disturbance of the conformation of gramicidin channels.

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The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited “molecular drill” oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.
The gramicidin ion channel: A model membrane protein
2007, Biochimica et Biophysica Acta - Biomembranes
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The natural gramicidin variants, gramicidin A, B, and C which differ at position 11 (see earlier) show distinctive channel properties [101]. In addition, photolysis of tryptophan by ultraviolet irradiation [102–105] or chemical modification by an oxidizing agent such as N-bromosuccinimide [106] leads to reduction in cation conductivity. Trp → Phe substitutions represent an interesting case since they are semi-conservative.
The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid–protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.
Sensitized Photoinactivation of Gramicidin Channels: Technique and Applications
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It has appeared that illumination with visible light in the presence of different photosensitizers effectively suppresses gramicidin channel activity. It should be noted that sensitized inactivation of gramicidin channels has much in common with another phenomenon discovered earlier – gramicidin inactivation by ultraviolet light in the absence of a photosensitizer [19–21]. Both processes are associated with damage to tryptophan residues, but only the sensitized photoinactivation involves participation of ROS.
This review is devoted to the analysis of sensitized photoinactivation of ionic channels formed by gramicidin A in bilayer lipid membranes. There has been a long way from the discovery of this phenomenon in 1992 and the first ideas concerning its mechanism to its application for studying the interaction of different agents with modified gramicidins. Measuring transmembrane current transients after a flash of visible light in the presence of a photosensitizer represents a very convenient method of determining rate constants of gramicidin channel formation and dissociation. In the present review, both the advantages and the drawbacks of the photoinactivation technique are compared with those of the conventional approaches, such as single-channel measurements, the voltage-jump technique, and the noise power spectrum analysis. A study of the interaction of charged gramicidin analogues with polyelectrolytes by using the sensitized photoinactivation procedure is described here in detail to demonstrate the wide potencies of this method. In addition, it is shown that the gramicidin channel photoinactivation can also be used for screening of new potent photosensitizers, and for the search of scavengers effectively protecting membrane systems from the attack of reactive oxygen species.
Peroxyl radicals promoted changes in water permeability through gramicidin channels in DPPC and lecithin-PC vesicles
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Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.
Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl choline large unilamellar vesicles
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In this work, we report the results bearing on the effect of gramicidin incorporation upon the dynamic and structural properties of dipalmitoyl phosphatidyl choline (DPPC) large unilamellar vesicle (LUVs). Results were obtained at different depths in the bilayer, both in the bulk of the bilayer and the vicinity of the added pentadecapeptide. This analysis was performed employing a series of extrinsic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan), pyrenedodecanoic acid, pyrene butyric acid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (Py–C₁₀–PC) and 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (bis-Py–C₁₀–PC)). Fluorescence measurements were carried out after direct excitation of the probe or after its excitation by resonant energy transfer (RET) from the intrinsic tryptophan groups of the polypeptide. The last procedure allows an evaluation of the lipidic annulus properties in the vicinity of the incorporated polypeptide.
In the gel state, gramicidin incorporation increases the order near the vesicle interface, as sensed by TMA-DPH. This increase is observed, above the phase transition temperature, over all the bilayer. This effect is probably related to a decrease in the lateral diffusion of the lipids, as evidenced by the reduced formation of intermolecular excimers following Py–C₁₀–PC excitation. Gramicidin incorporation also increases the penetration and/or mobility of water molecules located near the bilayer interface. In the gel phase, these changes are more relevant in the central part of the bilayer. When the LUVs are in the liquid crystalline state, the effect is observed through all the bilayer. In this state, the polypeptide incorporation also increases the diffusion/concentration of oxygen, irrespective of the probe localization.
Resonance energy transfer between Trp residues and Laurdan, Py–C₁₀–PC or bis-Py–C₁₀–PC dyes was employed to evaluate the properties of the lipidic annulus surrounding the polypeptide. In the gel phase, gramicidin incorporation produces an increase in the penetration and/or dynamics of water molecules, while in the liquid crystalline state it leads to a decrease of the acyl chain mobility in the deepest parts of the bilayer.
The monolayer technique: A potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes
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Erudites of the antiquity already knew the calming effect of oil films on the sea waves. But one had to wait until 1774 to read the first scientific report on oil films from B. Franklin and again 1878 to learn the thermodynamic analysis on adsorption developed by J. Gibbs. Then, in 1891, Agnes Pockels described a technique to manipulate oil films by using barriers. Finally, in 1917, I. Langmuir introduced the experimental and theoretical modern concepts on insoluble monolayers. Since that time, and because it has been found to provide invaluable information at the molecular scale, the monolayer technique has been more and more extensively used, and, during the past decade, an explosive increase in the number of publications has occurred. Over the same period, considerable and ever-increasing interest in the antimicrobial peptides of various plants, bacteria, insects, amphibians and mammals has grown. Because many of these antimicrobial peptides act at the cell membrane level, the monolayer technique is entirely suitable for studying their physicochemical and biological properties. This review describes monolayer experiments performed with some of these antimicrobial peptides, especially gramicidin A, melittin, cardiotoxins and defensin A. After giving a few basic notions of surface chemistry, the surface-active properties of these peptides and their behavior when they are arranged in monomolecular films are reported and discussed in relation to their tridimensional structure and their amphipathic character. The penetration of these antimicrobial peptides into phospholipid monolayer model membranes, as well as their interactions with lipids in mixed films, are also emphasized.

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Tryptophan photolysis is responsible for gramicidin-channel inactivation by ultraviolet light

Abstract

Biochim. Biophys. Acta

J. Mol. Biol.

J. Theor. Biol.

Biophys. Chem.

Biophys. J.

Pflugers Arch

J. Membrane Biol.

Annu. Rev. Physiol.

Biophys. J.