Bernard Desbat

Polarization Modulation FT-IR Spectroscopy of Surfaces and Ultra-Thin Films: Experimental Procedure and Quantitative Analysis:

Polarization modulation of the incident electromagnetic field is used to increase the sensitivity and the in situ experiment ability of the well-known method of reflexion absorption FT-IR spectroscopy, for the characterization of surfaces and ultra-thin films. The experimental procedure and signal processing of the detected intensity are described and illustrated with the use of results obtained with Langmuir-Blodgett monolayers. The quantitative analysis of the spectra is then developed, and a linear behavior of the band intensities is found for ultra-thin films exhibiting no strong absorptions. This result is checked with the use of organic and inorganic ultra-thin films of increasing thicknesses.

Polarization-modulated FT-IR spectroscopy of a spread monolayer at the air/water interface

This study devoted to the FT-IR spectroscopy of monolayers spread at the air/water interface is, to our knowledge, the first report presenting complete mid-infrared monolayer spectra perfectly extracted from the strong water vapor bands. This has been possible with the use of the polarization-modulated IRRAS method, which is not sensitive to the isotropic absorptions of the sample environment. On the basis of theoretical modeling and experiments, the best angle of incidence has been found near 76° for detection of intraplane as well as out-of-plane oriented monolayer absorptions. With the use of such experimental conditions, on the normalized difference (covered vs. uncovered water) PM-IRRAS spectra, monolayer vibrational bands come out upward or downward, depending on the orientation of their transition moment with respect to the interface. Application to the study of deuterated arachidic acid and arachidate monolayers allows observation of the vibrational modes of the polar head groups interacting with the liquid water molecules and provides some evidence of their symmetrical anchoring. The vibrational modes of the liquid water subphase contribute to these difference spectra as broad dips that certainly contain information on a possible restructuring of the water molecules at the interface.

Investigations at the air/water interface using polarization modulation IR spectroscopy

The ability of polarization modulation IR reflection absorption spectroscopy (PM–IRRAS) to study the air/water interface is presented. A brief description of the set-up and of the experimental procedure is given. Theoretical simulations accounting for the uniaxial nature of the spread monolayer lead to optimum experimental conditions (71 ° for the angle of incidence) and to a specific surface selection rule. Application to the study of cadmium arachidate, dimyristolyl phosphatidylcholine (DMPC) and polypeptidic Langmuir films illustrates the potential uses of this method.

Polarization modulation FTIR spectroscopy at the air-water interface

Abstract Mid-infrared spectra of monolayers spread at the air-water interface have been obtained, completely devoid of strong water vapor absorptions, using polarization modulation infrared reflexion absorption spectroscopy (PM-IRRAS). On normalized difference (covered vs. uncovered water) PM-IRRAS spectra, the monolayer absorption bands appear upwards or downwards depending on the orientation of their transition moment with respect to the water surface. Vibrational modes of the water subphase contribute to these difference spectra as broad dips. Study of a monolayer of cadmium arachidate has allowed observation of the vibrational modes of the polar heads and provides some evidence of their symmetrical anchoring at the water surface. Under surface compression, a monolayer of deuterated arachidic acid undergoes a molecular reorganization leading to a better ordering of the deuterated chains.

Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by ellipsometry and PM-IRRAS at the air–water interface

The penetratin peptide, a 16 amino acid sequence extracted from Antennapedia homeodomain, is able to translocate across a neural cell membrane through an unknown mechanism, most likely a non-specific interaction with membrane lipids. Beyond its potential application as vector targeting small hydrophilic molecules and enabling them to reach a cell nucleus, this observation raises intriguing questions concerning the physico-chemistry of peptide-lipid interactions. Here we present a study of the role of lipid surface pressure and head charge on the mechanism of interaction. This was performed using optical techniques: surface infrared spectroscopy and ellipsometry, applied to a monolayer of phospholipids deposited at the air-water interface. Determination of the structure and orientation of peptides and lipids (separately or together) evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption and its action on lipids.

Structure, orientation and affinity for interfaces and lipids of ideally amphipathic lytic LiKj(i=2j) peptides

The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.

Aggregation of Cateslytin β-Sheets on Negatively Charged Lipids Promotes Rigid Membrane Domains. A New Mode of Action for Antimicrobial Peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.

Ideally amphipathic β-sheeted peptides at interfaces: structure, orientation, affinities for lipids and hemolytic activity of (KL)mK peptides

Abstract Designed to model ideally amphipathic β-sheets, the minimalist linear (KL)mK peptides (m=4–7) were synthesized and proved to form stable films at the air/water interface, they insert into compressed dimyristoylphosphatidylcholine monolayers and interact with egg phosphatidylcholine vesicles. Whatever the interface or the lateral pressure applied to the films, FT-IR and polarization-modulated IRRAS spectroscopy developed in situ on the films indicated that all the peptides totally fold into intermolecular antiparallel β-sheets. Calculated spectra of the amide region allowed us to define the orientation of the β-strands compared to the interface. It is concluded that such β-sheets remain flat-oriented without deep perturbation of zwitterionic phospholipids. Dansyl labelling at the N-terminus indicates that all the peptides are monomeric at a low concentration in aqueous buffer and bind to lipids with similar Dns burying. The affinities for zwitterionic lecithin mono- and bilayers, quantitatively estimated from buffer to lipid partition constants, monotonically increased with peptide length, indicating that hydrophobicity is a limiting parameter for lipid and membrane affinities. Peptides induced permeability increases on zwitterionic liposomes, they are strongly hemolytic towards human erythrocytes and their activity increases concurrently with length. Taking into account the lipid affinity, a hemolytic efficiency can be defined: at the same amount of peptide bound, this efficiency strongly increases with the peptide length. It is proposed that the first determinant step of membrane disturbance is the invasion of the outer membrane leaflet by these ideally amphipathic β-sheeted structures lying flat at the interface, like large rafts depending on the number of β-strands.

Structure and denaturation of adsorbed lysozyme at the air–water interface

Adsorption of lysozyme at the surface of a buffer solution at 25 degrees C, pH 7, and ionic strength 0.1 is studied under different denaturing conditions using on X-ray reflectometry technique. When the lysozyme is fully denatured with urea and dithiothreitol (DTT), its measured adsorption profile is very well explained by the scale law (z(-4/3)) profile theoretically predicted for polymer adsorption. When no denaturing agent is present, a monolayer is also produced, but the adsorption profile cannot be explained by a monolayer of nondenatured lysozyme; furthermore, it is close to the one obtained for lysozyme partially denatured with urea. A PMIRRAS study of native lysozyme adsorbed at the air-buffer interface shows that the secondary structure of the protein is modified: most of the alpha-helices are replaced by beta-sheets. In contrast, when the lysozyme is adsorbed below a monolayer of oleic acid at the air-buffer interface, that is, on a hydrophilic interface, the protein forms a monolayer whose thickness, 3.0 nm, is equal to one dimension of crystallized lysozyme. Under such conditions, the adsorbed protein is not denatured. Thus the hydrophobic nature of the air-water interface yields partial denaturation of the protein upon adsorption, but the disulfur bridges and beta-sheets prevent total denaturation.

Quantitative Orientation Measurements in Thin Lipid Films by Attenuated Total Reflection Infrared Spectroscopy

Quantitative orientation measurements by attenuated total reflectance (ATR) infrared spectroscopy require the accurate knowledge of the dichroic ratio and of the mean-square electric fields along the three axes of the ATR crystal. In this paper, polarized ATR spectra of single supported bilayers of the phospholipid dimyristoylphosphatidic acid covered by either air or water have been recorded and the dichroic ratio of the bands due to the methylene stretching vibrations has been calculated. The mean-square electric field amplitudes were calculated using three formalisms, namely the Harrick thin film approximation, the two-phase approximation, and the thickness- and absorption-dependent one. The results show that for dry bilayers, the acyl chain tilt angle varies with the formalism used, while no significant variations are observed for the hydrated bilayers. To test the validity of the different formalisms, s- and p-polarized ATR spectra of a 40-A lipid layer were simulated for different acyl chain tilt angles. The results show that the thickness- and absorption-dependent formalism using the mean values of the electric fields over the film thickness gives the most accurate values of acyl chain tilt angle in dry lipid films. However, for lipid monolayers or bilayers, the tilt angle can be determined with an acceptable accuracy using the Harrick thin film approximation. Finally, this study shows clearly that the uncertainty on the determination of the tilt angle comes mostly from the experimental error on the dichroic ratio and from the knowledge of the refractive index.