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 streptavidin's fou…

Interaction between biotin lipids and streptavidin in monolayers: formation of oriented two-dimensional protein domains induced by surface recognition.

Highly specific ligand-receptor interactions generally characterize surface recognition reactions. Such processes can be simulated by streptavidin-biotin-specific binding. Biotin lipids have thus been synthesized, and their interaction with streptavidin (or avidin) at the air-water interface was directly shown by measurement of surface pressure isotherms and fluorescence microscopy. These proteins interact with the biotin lipid monolayer via specific binding or nonspecific adsorption. Both phenomena were clearly distinguished by use of the inactivated form of streptavidin. The binding of fluorescein-labeled streptavidin to monolayers was also directly observed by fluorescence microscopy. Th…

Ellipsometric study of the physical states of phosphatidylcholines at the air-water interface

Etude des couches monomoleculaires de dimyristoylphosphatidylcholine, de dipalmitoylphosphatidylcholine, de distearoyl phosphatidylcholine, de diarachidoylphosphatidylcholine a l'interface air/eau

Binding, Interaction, and Organization of Proteins with Lipid Model Membranes

Model membrane systems are used to investigate protein recognition and binding at interfaces. Fluorescence microscopy results are presented for interactions of the proteins, phospholipase A2 and antifluorescyl IgG, at lipid monolayer interfaces. Total internal reflection fluorescence measurements are used to quantify albumin and IgG adsorption to supported lipid monolayers.

Modeling of Cell Membrane Targeting: Specific Recognition, Binding, and Protein Domain Formation in Ligand-Containing Model Biomembranes

Drug delivery systems are designed to assist, accelerate, and control transport of pharmacologically active agents from sites of administration to specified targets in organs and tissues. So-called controlled drug delivery systems are intended to maintain continuously efficacious drug concentrations in vivo, either locally or systemically, over longer time periods. They should provide constant dosage levels above a minimum level of efficacy yet below mandated toxicity levels — a significant advantage over many conventional systemically administered formulations. Site-specific targeting of drugs, particularly those agents which prove highly toxic in small doses, can be utilized to maintain t…

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

AbstractPhospholipase 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 ext…

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 differe…

Surface properties of valine-gramicidin A at the air-water interface

Abstract Fluorescence microscopy, X-ray reflectivity and ellipsometry have been applied to the study of a monolayer of valine-gramicidin A (VGA) at the air-water interface to obtain insight into the conformation and orientation of VGA, and to clarify the shoulder observed in its π - A curve (12.5–16 mN m −1 , 240 – 175 A 2 molecule −1 ). At low molecular areas, the X-ray reflectivities are consistent with formation of a single layer of tubular-shaped dimers. Fluorescence microscopy yielded homogeneous pictures suggesting that on the macroscopic scale the shoulder cannot be attributed to domain formation. Thicknesses calculated from the ellipsometric isotherm are consistent with the X-ray da…

The binding of G-protein to rod outer segment phospholipids at the nitrogen–water interface

In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption–desorption in the monolayer of each main ROS phospholipi…

Ellipsometric and fluorescence microscopic investigations of a cyclam derivative at the air/water interface

Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations

Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4–4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interes…

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 …

Optical measurements of an acylated azacrown at the air-water interface

In this study, a cyclame derivative bearing four aliphatic chain substituents shows, like many amphiphiles, solid-condensed as well as liquid-expanded phases. In contrast to the classical amphiphiles, the onset of its phase transition is characterized by a bump-like shape whose amplitude is a function of the compression speed. Ellipsometry which is very sensitive to the monolayer physical state changes, and fluorescence microscopy which has contributed significantly to the understanding of the phenomena occurring in the phase transition region have been used to investigate the monolayer behavior of this compound. This study shows that in the liquid-expanded state, the film is homogeneous an…