Gerald Brezesinski

Langmuir monolayers to study interactions at model membrane surfaces

Langmuir monolayers at the liquid-air interface are well-defined interfacial systems and, therefore, excellent model systems to learn about interactions at interfaces beyond the classical DLVO description. Many parameters can be independently varied over a broad range and the structure can be analyzed with A precision. In the first part of the paper, the rich polymorphism in monolayers composed of amphiphilic molecules is demonstrated. Using homologues series generic phase diagrams can be derived. The delicate interplay of interactions causes a richness of phases which in turn can be used to measure fine variations in these interactions. Based on the understanding of the polymorphism in pure or mixed lipid monolayers, one can study the interaction of molecules dissolved in the subphase with monolayers. Samples presented are chemical reactions catalyzed by enzymes and coupling of polyelectrolytes to oppositely charged monolayers. To relate structure and reactivity, the activity of enzymes at the interface can be studied, predominantly combining X-ray diffraction and FTIR-spectroscopy. It is shown that the activity depends on monolayer structure. In one case, the reaction product leads to structural changes in the monolayer and stops the reaction, hence, indicating a subtle case of product inhibition via the membrane. On the other hand it has become possible to manipulate the organization of polyelectrolytes at interfaces via lipid charge density and ionic strength. In the most important case of DNA interacting with a membrane surface we show that DNA arranges at the interface in a lamellar manner, and the intermolecular distances, measured by Synchrotron X-ray diffraction can be varied by the lipid density.

Influence of ether linkages on the structure of double-chain phospholipid monolayers

Abstract The structure of phosphatidylcholine monolayers has been studied by Synchrotron X-ray diffraction at the air/water interface varying the ester and ether linkages of the aliphatic tails at the glycerol backbone. All systems investigated exhibit an oblique lattice structure with extremely large tilt angles of the chains from vertical, even at high lateral pressures. Although no large difference is seen in the isotherms, the replacement of ester by ether linkages causes a reduction of the tilt angle and of the area per molecule. These changes also depend on the position of the ether group with respect to the glycerol backbone and can be understood within a model where the carbonyl group of the ester at the C2 position pulls the attached chain towards the water subphase.

Adsorption of amyloid beta (1-40) peptide at phospholipid monolayers

The folding of amyloid β (1–40) peptide into β‐sheet‐containing fibrils is thought to play a causative role in Alzheimers disease. Because of its amphiphilic character, the peptide can interact with phospholipid membranes. Langmuir monolayers of negatively charged DPPS, DPPG, and DMPG, and also of zwitterionic DPPC and DMPC, have been used to study the influence of the peptide on the lipid packing and, vice versa, the influence of phospholipid monolayers on the peptide secondary structure by infrared reflection absorption spectroscopy and grazing incidence X‐ray diffraction. The peptide adsorbs at the air/water (buffer) interface, and also inserts into uncompressed phospholipid monolayers. When adsorbed at the interface, the peptide adopts a β‐sheet conformation, with the long axis of these β‐sheets oriented almost parallel to the surface. If the lipid exhibits a condensed monolayer phase, then compression of the complex monolayer with the inserted peptide leads to the squeezing out of the peptide at higher surface pressures (above 30 mN m−1). The peptide desorbs completely from zwitterionic monolayers and negatively charged DPPG and DPPS monolayers on buffer, but remains adsorbed in the β‐sheet conformation at negatively charged monolayers on water. This can be explained in terms of electrostatic interactions with the lipid head groups. It also remains adsorbed at, or penetrating into, disordered anionic monolayers on buffer. Additionally, the peptide does not influence the condensed monolayer structure at physiological pH and modest ionic strength.

Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide.

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimers disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-beta peptide (Abeta), the aggregating peptide in Alzheimers disease, is natively unfolded. The deposits found in Alzheimers disease are composed of Abeta fibrillar aggregates rich in beta-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Abeta peptide was studied by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce alpha-helix structure in Abeta, but their hydrogenated analogous lead to beta-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Abeta fibrillogenesis.

Langmuir monolayers as models to study processes at membrane surfaces

The use of new sophisticated and highly surface sensitive techniques as synchrotron based X-ray scattering techniques and in-house infrared reflection absorption spectroscopy (IRRAS) has revolutionized the monolayer research. Not only the determination of monolayer structures but also interactions between amphiphilic monolayers at the soft air/liquid interface and molecules dissolved in the subphase are important for many areas in material and life sciences. Monolayers are convenient quasi-two-dimensional model systems. This review focuses on interactions between amphiphilic molecules in binary and ternary mixtures as well as on interfacial interactions with interesting biomolecules dissolved in the subphase. The phase state of monolayers can be easily triggered at constant temperature by increasing the packing density of the lipids by compression. Simultaneously the monolayer structure changes are followed in situ by grazing incidence X-ray diffraction or IRRAS. The interactions can be indirectly determined by the observed structure changes. Additionally, the yield of enzymatic reaction can be quantitatively determined, secondary structures of peptides and proteins can be measured and compared with those observed in bulk. In this way, the influence of a confinement on the structural properties of biomolecules can be determined. The adsorption of DNA can be quantified as well as the competing adsorption of ions at charged interfaces. The influence of modified nanoparticles on model membranes can be clearly determined. In this review, the relevance and utility of Langmuir monolayers as suitable models to study physical and chemical interactions at membrane surfaces are clearly demonstrated.

Rationale for the design of shortened derivatives of the NK-lysin-derived antimicrobial peptide NK-2 with improved activity against Gram-negative pathogens.

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the α-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an α-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).

Binding of nonsteroidal anti-inflammatory drugs to DPPC: Structure and thermodynamic aspects

The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the phase transition and phase properties of 1,2-dipalmitoylphosphatidylcholine (DPPC) has been investigated in both 2D (monolayers at the air/water interface) and 3D (multilayers in lipid/water dispersions) model systems. The 2D membrane models have been characterized by means of pressure-area isotherms and grazing incidence X-ray diffraction (GIXD) measurements. Differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray diffraction have been applied to lipid aqueous dispersions. All NSAIDs studied altered the main transition temperature of the gel to liquid-crystalline phase transition, with the arylacetic acid derivatives (acemetacin and indomethacin) showing the largest effects. A comparison of the results reveals distinct structural features of the membrane models after interaction with the NSAID. All drugs induced perturbations in the lipid liquid-crystalline phase, suggesting a major change in the hydration behavior of DPPC. Again, the largest effects on the structural parameters are found for the arylacetic acid derivatives. The results obtained in the different model systems give indications of the role of the membrane/NSAID interactions that might also be important for NSAID gastric injury.

NSAIDs Interactions with Membranes: A Biophysical Approach

This work focuses on the interaction of four representative NSAIDs (nimesulide, indomethacin, meloxicam, and piroxicam) with different membrane models (liposomes, monolayers, and supported lipid bilayers), at different pH values, that mimic the pH conditions of normal (pH 7.4) and inflamed cells (pH 5.0). All models are composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which is a representative phospholipid of most cellular membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs in membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and penetration of NSAIDs into the membrane, IRRAS to acquire structural information of DPPC monolayers upon interaction with the drugs, and AFM to study the changes in surface topography of the lipid bilayers caused by the interaction with NSAIDs. The NSAIDs show pronounced interactions with the lipid membranes at both physiological and inflammatory conditions. Liposomes, monolayers, and supported lipid bilayers experiments allow the conclusion that the pH of the medium is an essential parameter when evaluating drug-membrane interactions, because it conditions the structure of the membrane and the ionization state of NSAIDs, thereby influencing the interactions between these drugs and the lipid membranes. The applied models and techniques provided detailed information about different aspects of the drug-membrane interaction offering valuable information to understand the effect of these drugs on their target membrane-associated enzymes and their side effects at the gastrointestinal level.

Specific adsorption of PLA2 at monolayers

Specific phospholipase A2 (PLA2) adsorption studies were performed on monolayers of d-dipalmitoyl-phosphatidylcholine (d-DPPC) and of an ether-ester d-1-O-hexadecyl-2-stearoyl-phosphatidylcholine (d-HSPC). In order to separate interfacial recognition from subsequent lipid cleavage, PLA2-resistant d-enantiomers were utilized for the investigations. Snake venom (N. naja naja and Crotalos atrox) PLA2, which hydrolyzes the sn-2 ester bond of l-phospholipids, was used. Fluorescence microscopy, film-balance pressure–area isotherms, and grazing incidence X-ray diffraction (GIXD) experiments were carried out. Fluorescence microscopy studies show that the enzyme accumulates preferentially at the liquid-expanded/condensed interface. At low surface pressure enzyme penetration into the monolayer is observed, whereas at high pressures the area per molecule is reduced upon specific adsorption. Monolayer structure, as determined by GIXD, is greatly affected by adsorption of PLA2. The tilt angle of the aliphatic chains of the monolayer becomes drastically reduced due to an enzyme-induced increase of the lipid packing efficiency. The unspecific adsorption of serum albumin to a d-DPPC monolayer does not change the monolayer structure. The structural changes, caused by PLA2 adsorption on d-enantiomer monolayers, are related to the chemical structure of the lipid molecules. Therefore, a relation between structure change and hydrolysis efficiency of PLA2 on the respective l-enantiomer monolayers can be assumed.

Nanotubes Protruding from Poly(allylamine hydrochloride)-Graft-Pyrene Microcapsules

Protrusion of one-dimensional nanotubes or nanorods from the poly(allylamine hydrochloride)-graft-pyrene (PAH-Py) microcapsules was discovered when the microcapsules were incubated in pH 0 and pH 2 solutions, respectively. Micelles assembled from deliberately synthesized PAH-Py polymers were also able to transform into one-dimensional structures, demonstrating the chemistry driven nature of the phenomenon. The one-dimensional nanotubes consisted of only 1-pyrenecarboxaldehyde with ordered π-π stacking, and showed a helical structure and anisotropic property. The hydrolysis of Schiff base and its rate at different pH values (10 times slower at pH 0 than at pH 2) played a key role in determining the final nanostructures, and the linear PAH directed the regular building up process especially for the nanotubes. Hollow capsules budded with nanotubes or nanorods mimicking the cellular protrusion of filopodia were successfully prepared by tuning the incubation pH and time. These results and the proposed mechanism open new opportunities for design of novel micronanostructures and materials for nanoscience, and biological and other advanced technologies.