Claude Lussan

The self-association of melittin and its binding to lipids: an intrinsic fluorescence polarization study.

Intrinsic fluorescence has proved to be very useful for studying protein-protein or lipid-protein interactions [l--8 J. Although these studies are mostly restricted to the analysis of intensity or wavelength changes in the emission spectra, polarization measurements can afford valuable information, as shown in the self-association of apo-lipoproteins [7,8] and the interaction of glucagon with lipids [5,6]. Here, intrinsic fluorescence polarization is applied to the study of the self-association of mellitin, and of its binding to lipids. Melittin is a small amphipathic peptide of 26 residues, extracted from bee venom, which is known to have a direct lytic activity on living cells [9-l 1 J. It contains only one fluorescent residue, Trp,,, and its emission spectrum is very sensitive to interactions with lipids [I ,3].

Variations in microviscosity values induced by different rotational behaviour of fluorescent probes in some aliphatic environments

Recently there have been large developments in the indirect estimation of phospholipid bilayer and membrane microviscosities through the use of fluorescent probes with the help of paraffin oils as standard solvents. Before applying this semi-empirical method to membrane systems, it seemed necessary to test: first, a large variety of probes (this has been done by many authors) and secondly, a large variety of aliphatic oils (there is little literature on these tests). The present paper shows the variations of the rotational relaxation rates of three probes in relation to the viscosities of some aliphatic oils. When changing the oil but keeping constant the macroscopic viscosity, large differences appear in the relaxation rates of a given fluorphore (a ratio of 30/1 is observed in the extreme cases). The microviscosities of membranes deduced from the probe motion will consequently exhibit large uncertainty, as is shown with dipalmitoyl phosphatidylcholine. The cause of these different behaviours must be looked for in the properties of the oil. Particularly, the anisotropy of the solute-solvent interaction in the site where the probe is located depends in part on the internal order of the solvent which is used as a reference.

Aliphatic chain transitions of phospholipid vesicles and phospholipid dispersions determined by polarization of fluoroscence

Abstract The polarization of fluorescence of dansyl phosphatidylethanolamine and 9-methylanthracene shows that these compounds are reliable indicators of the order-disorder transitions of the phospholipid aliphatic chains in bilayer systems. The transition is better defined in phospholipid dispersion than in vesicles. It is concluded that the two models are not identical as far as the structure near the melting temperature is concerned. Experiments in turbid solutions were performed with horizontal slits either in the incident or emitted beams, which eliminate the effect of light scattering. This improvement in experimental technique may facilitate the fluorescence polarization study of membrane suspensions.

Study of lipid—protein interactions in membrane models: Intrinsic fluorescence of cytochrome b5—phospholipid complexes

Until now, few integral membrane proteins have been isolated and studied; cytochrome h5 is one of them. Strittmatter et al. [I ] and Ememoto and Sato [2] first demonstrated that pure detergent extracted cytochrome h5 can bind to microsomes. Furthermore, Sullivan and Holloway [3] showed that this protein interacts with egg phosphatidylcholine. By reconstitution experiments with single shell vesicles? Dufourcq et al. [4] and Robinson and Tanford [S] obtained a more detailed knowledge of the lipid-protein complexes. Cytochrome b5 can be described as a protein composed of two different moieties: one. bearing the heme, is hydrophilic and can be obtained by trypsin action on microsomes (cytochrome t-b,). The second one is composed of about fifty four residues for bovine liver extracted protein, and would be responsible for the binding of the whole protein to the membrane [6,7]. Furthermore, this hydrophobic peptide contains several aromatic residues; four of them are tryptophan residues [8]. Fluorescence is a classical method to study the environment of aromatic residues in soluble proteins [9,10]. This technique has also been used to look at interactions between apolipoproteins and lipids [11,12,13]. In this paper, we report variations of the fluorescence parameters during interactions of purified bovine liver cytochrome b5 with selected phospholipids, in order to obtain information on the structure of the lipidprotein complexes as an approach to the structure of membranes. 2. Materials and methods

Binding of bovine cytochrome b5 to phosphatidycholine liposomes Characterization of the reconstituted lipid-protein vesicles

Abstract Cytochrome b 5 was extracted and purified from beef liver by a detergent method (cytochrome d-b 5 ). The hydrophilic moiety which carries the heme group (cytochrome t-b 5 ) was prepared by trypsin action upon pure cytochrome d-b 5 . Single-shelled lecithin liposomes form complexes with cytochromes d-b 5 up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [3H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes. Cytochrome t-b 5 does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents. Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature T M . i.e. when the aliphatic chains are in a crystalline state, and above T M , when the alipathic chain are in a fluid state. 1H NMR spectra show that even at the maximum cytochrome d-b 5 concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex.

Lipid-protein interactions in membrane models. Fluorescence polarization study of cytochrome b5-phospholipids complexes.

According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5. Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very sensitive to the binding of proteins, and so is a useful method to study lipid-protein interactions. The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of bilayer, only due to the interaction of the hydrophobic peptide tail. Interaction of dipalmitoly phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 degrees C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected. Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.

Effects of ions on vesicles and phospholipid dispersions studied by polarization of fluorescence

Abstract 1. 1. The oxidation of egg lecithin leads to an increase in the degree of polarization of the fluorescent probe dansylphosphatidylethanolamine. This effect can be avoided by working under nitrogen. 2. 2. The binding of cations on phospholipid dispersions or phospholipid vesicles significantly enhances the degree of polarization of the fluorescent probe dansylphosphatidylethanolamine. This enhancement is attributed to an increase in order of the phospholipid leaflet. 3. 3. The observed effect is proportional to the negative charge of vesicles or to the positive charge of ions (Na + 2+ 3+ ). 4. 4. The temperature of the order-disorder transition of the aliphatic chains of the dimyristoylphosphatidylcholine caused by La 3+ is 3 °C. 5. 5. The kinetic study of the binding of La 3+ on vesicles indicates that the cations do not diffuse through the bilayer of pure egg lecithin. However, in the case of vesicles charged with phosphatidic acid, the observed evolution can be explained by diffusion of the La 3+ through the bilayer and binding on the internal layer of the vesicles, or by a rupture of the vesicles.

Mean viscosities in microscopic systems and membrane bilayers: a semi-empirical general basis applicable to different kinds of extrinsic probes.

Severe limitations to semi-empirical uses of StokesEinstein relation for estimations of microviscosities in membranes have recently arisen. Studies on the motions of a rod-like fluorescent molecule by the timeresolved components of its polarized light emission [l-6] cast some doubt on the previous estimations. When a probe of this shape is inserted in a bilayer and submitted to a pulsed excitation, a stationary light anisotropy remains at long times; this effect lacks in homogeneous viscous liquids which have been studied until now and probably reflects a hindrance of rotation. This restriction must be looked for in the peculiar space order of the lipidic chains. We have shown [7] that when studying a wide range of viscous liquids by combining steady state polarization and life time measurements of the precedent probe, the method [8-IO] was unable to predict even the viscosity of an oil from those of the others: with still more reason, a mean viscosity in membranes seemed difficult to be reached by this way. However, the different behavior of the rod-like probe in homogeneous liquids is also set up on the various local orders and this situation is not actually different from the comparison between one of the liquids and a bilayer. In fact the problem is to determine if an average viscosity may still have any meaning inside the bilayer, despite the transversal order gradient which has been longknown [11,12]. We further experimented with such systems of various viscosities and local order: we can now pro-

Use of the radical anion TCNQ.̄ as a spin probe of the polar heads transition in some phospholipids

Biological membranes are constituted of lipids and proteins. Their general molecular organization is now well known: phospholipid bimolecular leaflet in which proteins are more or less embedded [1,2]. Important results have been obtained on the structure of phospholipid aliphatic chains principally on their order-disorder transition by differential scanning calorimetry [3] fluorescent [4] and spin-labelled probes [5]. But the structure and conformational changes of the membrane-water interface constituted mainly of the phospholipid polar head groups is not entirely understood. Recently nmr studies of phosphorus [6] and deuterated choline moiety [7] showed that transition of phospholipid polar heads occurs. In this paper we report preliminary results obtained with a new ESR probe, the tetracyanoquinodimethane (TCNQ) [8]. This molecule is a strong n acid and leads to charge transfer complexes with a variety of Lewis bases; with the quaternary ammonium functions a stable radical anion TCNQ= is formed [9].

Construction et mise au point d’un appareil de mesure de la polarisation de fluorescence. Exemple d’application à l’étude de modèles de membranes biologiques

Dans ce memoire est decrit un appareil de mesure de ia polarisation de fluorescence, qui permet la determination quasi simultanee des rapports I // /I 0 et [math] des intensites des deux composantes de la lumiere emise sur l’intensite d’un faisceau de reference. Grâce a ce dispositif, qui assure la stabilite du signal au cours du temps, et a la moyennisation automatique d’un grand nombre de mesures, il est possible d’obtenir avec une tres bonne precision le taux de polarisation P, (ΔP ≤ 0,003) et l’intensite totale ([math]) de la lumiere emise par un echantillon. De plus, l’uLilisation de cet appareil pour des etudes cinetiques peut etre envisagee.Un exemple d’application de la polarisation de fluorescence aux systemes modeles de membranes biologiques est ensuite donne, par la mise en evidence, au moyen du 1,6-diphenyl- 1,3,5-hexatriene, des transitions de phase de la dipalmitoyl- phosphatidylcholine et de la dimyristoylphosphatidylcholine.