Christian Salesse

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

An enzyme caught in action: Direct imaging of hydrolytic function and domain formation of phospholipase A2 in phosphatidylcholine monolayers

Phospholipase A2, a ubiquitous lipolytic enzyme that actively catalyses hydrolysis of phospholipids, has been studied as a model for enzyme‐substrate reactions, as a membrane structural probe, and as a model for lipid‐protein interactions. Its mechanism of action remains largely controversial. We report here for the first time direct microscopic observation of the lipolytic action of fluorescently marked phospholipase A2 (Naja naja naja) against phosphatidylcholine monolayers in the lipid phase transition region. Under these conditions, phospholipase A2 is shown to target and hydrolyse solid‐phase lipid domains of L‐α‐dipalmitoylphosphatidylcholine. In addition, after a critical extent of monolayer hydrolysis, the enzyme itself aggregates into regular, visible proteinaceous domains within the lipid monolayer. Solid‐phase lipid hydrolysis indicates a preferential hydrolytic environment for phospholipase A2 while enzyme domain formation points to a possible allosteric inhibition mechanism by hydrolysis products.

Specific recognition and formation of two- dimensional streptavidin domains in monolayers: applications to molecular devices

Abstract By virtue of the high-affinity specific interaction between the vitamin, biotin, and the protein, streptavidin, monolayers of synthetic lipids with biotin headgroups can tightly bind streptavidin at the lipid-water interface. Through this specific recognition fluorescently-labelled streptavidin spontaneously organizes in the plane of the interface to form large protein domains, directly visible in situ by fluorescence microscopy and exhibiting optical anisotropy. Further structural characterization has shown that these domains are two-dimensional protein crystals. Correlation with the known three-dimensional crystal structure of streptavidin indicates that two of streptavidins four biotin binding sites are free for further derivatization to create multilayered organized protein molecular devices.

Polarization-Modulated Infrared Spectroscopy and X-Ray Reflectivity of Photosystem II Core Complex at the Gas-Water Interface

The state of photosystem II core complex (PS II CC) in monolayer at the gas-water interface was investigated using in situ polarization-modulated infrared reflection absorption spectroscopy and x-ray reflectivity techniques. Two approaches for preparing and manipulating the monolayers were examined and compared. In the first, PS II CC was compressed immediately after spreading at an initial surface pressure of 5.7 mN/m, whereas in the second, the monolayer was incubated for 30 min at an initial surface pressure of 0.6 mN/m before compression. In the first approach, the protein complex maintained its native alpha-helical conformation upon compression, and the secondary structure of PS II CC was found to be stable for 2 h. The second approach resulted in films showing stable surface pressure below 30 mN/m and the presence of large amounts of beta-sheets, which indicated denaturation of PS II CC. Above 30 mN/m, those films suffered surface pressure instability, which had to be compensated by continuous compression. This instability was correlated with the formation of new alpha-helices in the film. Measurements at 4 degreesC strongly reduced denaturation of PS II CC. The x-ray reflectivity studies indicated that the spread film consists of a single protein layer at the gas-water interface. Altogether, this study provides direct structural and molecular information on membrane proteins when spread in monolayers at the gas-water interface.

Mixed monolayers of natural and polymeric phospholipids: structural characterization by physical and enzymatic methods

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

An evaluation of purity criteria for bovine rod outer segment membranes

The purity criteria of bovine rod outer segments (ROS) purified by different procedures were evaluated. Bovine ROS were purified by flotation and/or sedimentation in a continuous concentration gradient of sucrose. The purity of the different fractions was then evaluated according to four purity criteria: (i) the A280/delta A500 ratio, (ii) the moles of phospholipid per mole of rhodopsin, (iii) the fatty acid composition, and (iv) the interfacial properties of ROS membranes. All the purity criteria, except the A280/delta A500 ratio, were found to be adequate. From our results, the A280/delta A500 ratio cannot be used alone to characterize ROS purity. Furthermore, the phospholipid-to-rhodopsin ratio appears as the best purity criterion because of its reliability, its higher sensitivity, and its ease of achievement. It is noteworthy that mechanical treatment of the retinas dramatically affects the purification of ROS.

Ellipsometric studies of rod outer segment phospholipids at the nitrogen-water interface☆

Abstract Ellipsometry is an optical method which uses the principle that the state of polarization of polarized light changes on reflection at an interface. We have constructed a null ellipsometer for the study of the optical properties of monomolecular films of biological interest at the nitrogen-water interface. With this apparatus we have reproduced the known ellipsometric properties of arachidic acid spread on acidic aqueous solution. Then, we have performed surface potential, surface pressure and ellipsometric measurements of PC (18:1) and rod outer segment phospholipids, i.e. PE ros , PC ros and PS ros , at the nitrogen-water interface. Based on literature refractive indices data, results provide information about the thickness of a lipid film in the close-packed region. Pertinent information about molecular organization of the film is deduced from the close relationship observed between surface potential and ellipsometric isotherms. The importance of these results on the discal membrane structure and function is discussed.

Monitoring of phospholipid monolayer hydrolysis by phospholipase A2 by use of polarization-modulated Fourier transform infrared spectroscopy

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) was used to follow the hydrolysis of phospholipid monolayers at the air-water interface by phospholipase A2 (PLA2). The decrease in the intensity of the nuC=O ester band of dipalmitoylphosphatidylcholine at 1733 cm(-1) and the appearance of two new infrared bands in the 1530-1580 cm(-1) region allowed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in the intensity of the band at 1733 cm(-1) was attributed to the enzymatic hydrolysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol backbone whereas the doublet appearing at 1537 and 1575 cm(-1) was attributed to the nu(a) COO- vibration of the newly formed calcium-palmitate. The presence of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously postulated mechanism for the formation of PLA2 domains at the air-water interface. Definitive assignment of the infrared bands has been possible by measuring PM-IRRAS spectra of the individual hydrolysis products (palmitic acid and lysopalmitoylphosphatidylcholine) as well as of 1-caproyl-2-palmitoyl-phosphatidylcholine and 1-palmitoyl-2-caproylphosphatidylcholine monolayers before and after hydrolysis by PLA2.

The behavior of membrane proteins in monolayers at the gas-water interface : comparison between photosystem II, rhodopsin and bacteriorhodopsin

Abstract It has been postulated, without supporting evidence, for decades that proteins are denatured once spread in monolayers at the gas–water interface. In the present study, the effect of different experimental conditions on the structure of three membrane proteins has been investigated by polarization modulated infrared reflection absorption spectroscopy in situ in monolayers at the gas–water interface. We have found that photosystem II core complex (PS II CC) is less sensitive to denaturation than rhodopsin. In fact, denaturation of rhodopsin could only be prevented when spreading was performed at 4°C. In contrast, bacteriorhodopsin was found to remain native when monolayer spreading was performed in conditions that were found to denature both PS II CC and rhodopsin. This behavior may be explained by the two-dimensional crystalline structure of bacteriorhodopsin. In conclusion, conditions can be found where the native structure of membrane proteins is maintained after their spreading in monolayers at the gas–water interface.

eSpectroscopic and Structural Properties of Valine Gramicidin A in Monolayers at the Air-Water Interface

Monomolecular films of valine gramicidin A (VGA) were investigated in situ at the air-water interface by x-ray reflectivity and x-ray grazing incidence diffraction as well as polarization modulation infrared reflection absorption spectros- copy (PM-IRRAS). These techniques were combined to obtain information on the secondary structure and the orientation of VGA and to characterize the shoulder observed in its -A isotherm. The thickness of the film was obtained by x-ray reflectivity, and the secondary structure of VGA was monitored using the frequency position of the amide I band. The PM-IRRAS spectra were compared with the simulated ones to identify the conformation adopted by VGA in monolayer. At large molecular area, VGA shows a disordered secondary structure, whereas at smaller molecular areas, VGA adopts an anti-parallel double-strand intertwined 5.6 helical conformation with 30° orientation with respect to the normal with a thickness of 25 A. The interface between bulk water and the VGA monolayer was investigated by x-ray reflectivity as well as by comparing the experimental and the simulated PM-IRRAS spectra on D2O and H2O, which suggested the presence of oriented water molecules between the bulk and the monolayer.