W. Urbach

Hydration and protein folding in water and in reverse micelles: compressibility and volume changes.

The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies.

Phospholipid decoration of microcapsules containing perfluorooctyl bromide used as ultrasound contrast agents

We present here an easy method to modify the surface chemistry of polymeric microcapsules of perfluorooctyl bromide used as ultrasound contrast agents (UCAs). Capsules were obtained by a solvent emulsification-evaporation process with phospholipids incorporated in the organic phase before emulsification. Several phospholipids were reviewed: fluorescent, pegylated and biotinylated phospholipids. The influence of phospholipid concentration on microcapsule size and morphology was evaluated. Only a fraction of the phospholipids is associated to microcapsules, the rest being dissolved with the surfactant in the aqueous phase. Microscopy shows that phospholipids are present within the shell and that the core/shell structure is preserved up to 0.5 mg fluorescent phospholipids, up to about 0.25 mg pegylated phospholipids or biotinylated phospholipids (for 100 mg of polymer, poly(lactide-co-glycolide) (PLGA)). HPLC allows quantifying phospholipids associated to capsules: they correspond to 10% of pegylated phospholipids introduced in the organic phase. The presence of pegylated lipids at the surface of capsules was confirmed by X-ray photon electron spectroscopy (XPS). The pegylation did not modify the echographic signal arising from capsules. Finally biotinylated microcapsules incubated with neutravidin tend to aggregate, which confirms the presence of biotin at the surface. These results are encouraging and future work will consist of nanocapsule surface modification for molecular imaging.

Echography using correlation techniques: choice of coding signal

Theoretical studies made in the early 1980s suggest that ultrasonic imaging using correlation technique can overcome some of the drawbacks of classical pulse echography. Indeed by transmitting a continuous coded signal and then compressing it into a short, high resolution pulse at the receiver the total signal to noise ratio (SNR) is improved. The target location is determined by cross correlation of the emitted and the received signal. The band compression allows, by increasing SNR, the retrieval of echo signals buried in the receiver noise. Thus in medical-type echography, where the signal attenuation at fixed depth is proportional to the frequency, the SNR improvement allows the use of higher frequency signals and leads to improved resolution. We report here the results of comparative experimental studies of simple echo B type images as obtained by the classical pulse echo and correlation techniques. Because the optimisation of the coded signal plays a crucial role in the performance of the correlation technique we will also present a comparative study of the performances of the most common codes (m-sequences and complementary series). In particular we shall emphasise the following points: the relative importance of the central lobe as compared to the side lobes of the correlation function, which is directly related to the dynamic of the imaging system, the width of the correlation peak which is directly related to the axial resolution of the system, the facility of the realisation. The merit of B-mode images obtained with the coded signals will be discussed showing that as far as signal modulation is used the best results are obtained with periodic m-sequences.<<ETX>>

Proteins in membrane mimetic systems. Insertion of myelin basic protein into microemulsion droplets

The insertion of myelin basic protein into microemulsion droplets of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) has been studied by quasi-elastic light scattering. Measurements were made at both low and high molar ratios of water to surfactant, as a function of protein occupancy. The hydrodynamic radii of filled and empty droplets were experimentally evaluated. These were compared to values calculated using a water shell model of protein encapsulation, and excellent agreement was obtained. At low molar ratio of water to surfactant (w0 = 5.6), the hydrodynamic radius of filled droplets is significantly larger than the radius of empty ones. Under these conditions, about three empty (water-filled) droplets are required to build up a droplet of sufficient size to accommodate a single protein molecule. At maximum solubilization, which occurs at w0 = 5.6, a small fraction of droplets are found containing protein aggregates. In contrast, results at high values of w0 (22.4) reveal radii for empty and occupied droplets of comparable dimension, and the absence of aggregates. The results are discussed in terms of the model and the mechanism of interaction of this protein with the aqueous interfaces provided by these membrane-mimetic systems.

Recent applications of fluorescence recovery after photobleaching (FRAP) to membrane bio-macromolecules.

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods.

Tracking membrane protein association in model membranes.

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5. Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.

Self‐diffusion of interacting micelles: FRAPP study of micelles self‐diffusion

We have studied self‐diffusion in DTAB micellar systems at three different salinities with fluorescence photobleaching experiments. In order to characterize interactions in these systems, we have also performed light scattering experiments. Despite of the strong variation of the interactions with salinities, almost no salinity variation in self‐diffusion coefficients vs droplets volume fraction curves has been observed. A clear difference between the friction coefficients involved in self‐ and mutual diffusion have been evidenced.

Structural parameters of the myelin transmembrane proteolipid in reverse micelles

The Folch-Pi proteolipid is the most abundant structural protein from the central nervous system myelin. This protein-lipid complex, normally insoluble in water, requires only a small amount of water for solubilization in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The characterization of the proteolipid-free and proteolipid-containing micelles was undertaken by light scattering and fluorescence recovery after fringe pattern photobleaching (FRAPP) experiments. Quasi elastic light scattering (QELS) was carried out at a high (200 mM) AOT concentration, at low water-to-surfactant mole ratio (Wo = 7) and at increasing protein occupancy. Two apparent hydrodynamic radii, differing tenfold in size, were obtained from correlation functions. The smaller one (RaH = 5.2 nm) remains constant and corresponds to that measured for protein-free micelles. The larger one increases linearly with protein concentration. In contrast, FRAPP measurements of self-diffusion coefficients were found unaffected by the proteolipid concentration. Accordingly, they have been performed at constant protein/surfactant mole ratios. The equivalent RH, extrapolated to zero AOT concentration for protein-free reverse micelles (2.9 nm) and in the presence of the proteolipid (4.6 nm), do not reveal the mode of organization previously suggested by QELS measurements. The complex picture emerging from this work represents a first step in the characterization of an integral membrane protein in reverse micelles.

A self-diffusion study of polymer-like micelles

The authors present a self-diffusion study of long cylindrical micelles of lecithin in an organic solvent. The results are compared with those obtained for similar micelles of CTAB (cethyltrimethylammonium bromide) in brine. Qualitatively they observe a similar behaviour for both systems. The measured self-diffusion coefficient Ds has a power law dependence on the surfactant concentration. However, the exponent, which is salinity-dependent in the CTAB system, is constant and in good agreement with a theory for living polymers in the lecithin system. In the case of very elongated lecithin and CTAB micelles, Ds becomes independent of surfactant concentration.

Ultrasonic Studies of Alcohol-Induced Transconformation in β-Lactoglobulin: The Intermediate State

In mixed alcohol-water solvents, bovine beta-lactoglobulin undergoes a cooperative transition from beta-sheet to a high alpha-helix content conformer. We report here the characterization of beta-lactoglobulin by compressibility and spectroscopy measurements during this transconformation. Both the volume and compressibility increase as a function of alcohol concentration, up to maximal values which depend on the chemical nature of the three alcohols used: hexafluoroisopropanol, trifluoroethanol, and isopropanol. The order of effectiveness of alcohols in inducing the compressibility transition is identical to that previously reported for circular dichroism and thus independent of the observation technique. The highly cooperative sigmoidal curves found by compressibility determination match closely those obtained by circular dichroism at 222 nm, indicating a correlation between the two phenomena measured by the two different techniques. The presence of an equilibrium intermediate form was shown by the interaction of beta-lactoglobulin with 8-anilino-1-naphthalene sulfonic acid, a probe widely used to detect molten-globule states of proteins. It was correlated with the plateau region of the volume curves and with the inflexion points of the sigmoidal compressibility curves. Ultrasound characterization of proteins can be carried out in optically transparent or nontransparent media.