Élodie Boisselier

Analysis of the Contribution of Saturated and Polyunsaturated Phospholipid Monolayers to the Binding of Proteins

The binding of peripheral proteins to membranes results in different biological effects. The large diversity of membrane lipids is thought to modulate the activity of these proteins. However, information on the selective binding of peripheral proteins to membrane lipids is still largely lacking. Lipid monolayers at the air/water interface are useful model membrane systems for studying the parameters responsible for peripheral protein membrane binding. We have thus measured the maximum insertion pressure (MIP) of two proteins from the photoreceptors, Retinitis pigmentosa 2 (RP2) and recoverin, to estimate their binding to lipid monolayers. Photoreceptor membranes have the unique characteristic that more than 60% of their fatty acids are polyunsaturated, making them the most unsaturated natural membranes known to date. These membranes are also thought to contain significant amounts of saturated phospholipids. MIPs of RP2 and recoverin have thus been measured in the presence of saturated and polyunsaturated phospholipids. MIPs higher than the estimated lateral pressure of biomembranes have been obtained only with a saturated phospholipid for RP2 and with a polyunsaturated phospholipid for recoverin. A new approach was then devised to analyze these data properly. In particular, a parameter called the synergy factor allowed us to highlight the specificity of RP2 for saturated phospholipids and recoverin for polyunsaturated phospholipids as well as to demonstrate clearly the preference of RP2 for saturated phospholipids that are known to be located in microdomains.

Influence of the physical state of phospholipid monolayers on protein binding.

Langmuir monolayers were used to characterize the influence of the physical state of phospholipid monolayers on the binding of protein Retinis Pigmentosa 2 (RP2). The binding parameters of RP2 (maximum insertion pressure (MIP), synergy and ΔΠ(0)) in monolayers were thus analyzed in the presence of phospholipids bearing increasing fatty acyl chain lengths at temperatures where their liquid-expanded (LE), liquid-condensed (LC), or solid-condensed (SC) states can be individually observed. The data show that a larger value of synergy is observed in the LC/SC states than in the LE state, independent of the fatty acyl chain length of phospholipids. Moreover, both the MIP and the ΔΠ(0) increase with the fatty acyl chain length when phospholipids are in the LC/SC state, whereas those binding parameters remain almost unchanged when phospholipids are in the LE state. This effect of the phospholipid physical state on the binding of RP2 was further demonstrated by measurements performed in the presence of a phospholipid monolayer showing a phase transition from the LE to the LC state at room temperature. The data collected are showing that very similar values of MIP but very different values of synergy and ΔΠ(0) are obtained in the LE (below the phase transition) and LC (above the phase transition) states. In addition, the binding parameters of RP2 in the LE (below the phase transition) as well as in the LC (above the phase transition) states were found to be indistinguishable from those where single LC and LE states are respectively observed. The preference of RP2 for binding phospholipids in the LC state was then confirmed by the observation of a large modification of the shape of the LC domains in the phase transition. Therefore, protein binding parameters can be strongly influenced by the physical state of phospholipid monolayers. Moreover, measurements performed with the α/β domain of RP2 strongly suggest that the β helix of RP2 plays a major role in the preferential binding of this protein to phospholipids in the LC state.

Effect of oxidation of polyunsaturated phospholipids on the binding of proteins in monolayers

Polyunsaturated fatty acids (PUFA) are particularly susceptible to oxidation. The resulting oxidized products may exert toxic effects. In particular, information is lacking on the effect of oxidized polyunsaturated phospholipid membranes on protein binding. This is particularly important for photoreceptors where many processes take place at the membrane surface because of their very large content in polyunsaturated phospholipids. Langmuir monolayers were thus used to determine the effect of oxidized phospholipids on the binding parameters of two proteins located in photoreceptors: Retinitis pigmentosa 2 (RP2) and recoverin. Measurements were performed using lipid oxidized during storage in solution and directly at the air-water interface. Large differences were observed between the binding parameters of RP2 and recoverin in the presence of intact and oxidized 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (DDPC). Indeed, large decreases of the maximum insertion pressure, ΔΠ0 and ΔΠ were observed when protein binding was compared between intact and oxidized DDPC. Altered protein binding in the presence of oxidized lipids could thus lead to improper membrane processes and various cellular malfunctioning and diseases.

How to gather useful and valuable information from protein binding measurements using Langmuir lipid monolayers

This review presents data on the influence of various experimental parameters on the binding of proteins onto Langmuir lipid monolayers. The users of the Langmuir methodology are often unaware of the importance of choosing appropriate experimental conditions to validate the data acquired with this method. The protein Retinitis pigmentosa 2 (RP2) has been used throughout this review to illustrate the influence of these experimental parameters on the data gathered with Langmuir monolayers. The methods detailed in this review include the determination of protein binding parameters from the measurement of adsorption isotherms, infrared spectra of the protein in solution and in monolayers, ellipsometric isotherms and fluorescence micrographs.

Towards a Multifunctional Electrochemical Sensing and Niosome Generation Lab-on-Chip Platform Based on a Plug-and-Play Concept

In this paper, we present a new modular lab on a chip design for multimodal neurotransmitter (NT) sensing and niosome generation based on a plug-and-play concept. This architecture is a first step toward an automated platform for an automated modulation of neurotransmitter concentration to understand and/or treat neurodegenerative diseases. A modular approach has been adopted in order to handle measurement or drug delivery or both measurement and drug delivery simultaneously. The system is composed of three fully independent modules: three-channel peristaltic micropumping system, a three-channel potentiostat and a multi-unit microfluidic system composed of pseudo-Y and cross-shape channels containing a miniature electrode array. The system was wirelessly controlled by a computer interface. The system is compact, with all the microfluidic and sensing components packaged in a 5 cm × 4 cm × 4 cm box. Applied to serotonin, a linear calibration curve down to 0.125 mM, with a limit of detection of 31 μM was collected at unfunctionalized electrodes. Added sensitivity and selectivity was achieved by incorporating functionalized electrodes for dopamine sensing. Electrode functionalization was achieved with gold nanoparticles and using DNA and o-phenylene diamine polymer. The as-configured platform is demonstrated as a central component toward an “intelligent” drug delivery system based on a feedback loop to monitor drug delivery.

Lipid Selectivity, Orientation, and Extent of Membrane Binding of Nonacylated RP2.

Retinitis pigmentosa 2 (RP2) is an ubiquitary protein of 350 residues. The N-terminus of RP2 contains putative sites of myristoylation and palmitoylation. The dually acylated protein is predominantly localized to the plasma membrane. However, clinically occurring substitution mutations of RP2 in photoreceptors lead to the expression of a nonacylated protein, which was shown to be misrouted to intracellular organelles using different cell lines. However, the parameters responsible for the modulation of the membrane binding of nonacylated RP2 (naRP2) are still largely unknown. The maximal insertion pressure of naRP2 has thus been determined after its injection into the subphase underneath monolayers of phospholipids, which are typical of photoreceptor membranes. These data demonstrated that naRP2 shows a preferential binding to saturated phospholipid monolayers. Moreover, polarization modulation infrared reflection absorption spectroscopy has allowed comparison of the secondary structure of this protein in solution and upon binding to phospholipid monolayers. In addition, simulations of these spectra have allowed to determine that the β-helix of naRP2 has an orientation of 60° with respect to the normal, which remains unchanged regardless of the type of phospholipid. Finally, ellipsometric measurements of naRP2 demonstrated that its particular affinity for saturated phospholipids can be explained by its larger extent of insertion in this phospholipid monolayer compared to that in polyunsaturated phospholipid monolayers.

Membrane binding of Neuronal Calcium Sensor-1 (NCS1).

Neuronal Calcium Sensor-1 (NCS1) belongs to the family of Neuronal Calcium Sensor (NCS) proteins. NCS1 is composed of four EF-hand motifs and an N-terminal myristoylation. However, the presence of a calcium-myristoyl switch in NCS1 and its role in the membrane binding are controversial. The model of Langmuir lipid monolayers is thus used to mimic the cell membrane in order to characterize the membrane interactions of NCS1. Two binding parameters are calculated from monolayer measurements: the maximum insertion pressure, up to which protein binding is energetically favorable, and the synergy, reporting attractive or repulsive interactions with the lipid monolayers. Binding membrane measurements performed in the presence of myristoylated NCS1 reveal better binding interactions for phospholipids composed of phosphoethanolamine polar head groups and unsaturated fatty acyl chains. In the absence of calcium, the membrane binding measurements are drastically modified and suggest that the protein is more strongly bound to the membrane. Indeed, the binding of calcium by three EF-hand motifs of NCS1 leads to a conformation change. NCS1 arrangement at the membrane could thus be reshuffled for better interactions with its substrates. The N-terminal peptide of NCS1 is composed of two amphiphilic helices involved in the membrane interactions of NCS1. Moreover, the presence of the myristoyl group has a weak influence on the membrane binding of NCS1 suggesting the absence of a calcium-myristoyl switch mechanism in this protein. The myristoylation could thus have a structural role required in the folding/unfolding of NCS1 which is essential to its multiple biological functions.

A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate

Glutathione S-transferase (GST) is widely used to prepare and purify GSTtagged fusion proteins. Although GST improves protein solubility, detergents must often be used to achieve protein solubilization from bacterial lysates. However, purification of GST by affinity chromatography cannot be achieved in the presence of even low concentrations of the detergent sodium dodecyl sulfate (SDS). Here we show that 2-methyl-2,4-pentanediol (MPD) can prevent SDS from interfering with purification of GST, thus enabling purification of proteins that require SDS to improve their solubility.

Miniaturized FDDA and CMOS Based Potentiostat for Bio-Applications

A novel fully differential difference CMOS potentiostat suitable for neurotransmitter sensing is presented. The described architecture relies on a fully differential difference amplifier (FDDA) circuit to detect a wide range of reduction-oxidation currents, while exhibiting low-power consumption and low-noise operation. This is made possible thanks to the fully differential feature of the FDDA, which allows to increase the source voltage swing without the need for additional dedicated circuitry. The FDDA also reduces the number of amplifiers and passive elements in the potentiostat design, which lowers the overall power consumption and noise. The proposed potentiostat was fabricated in 0.18 µm CMOS, with 1.8 V supply voltage. The device achieved 5 µA sensitivity and 0.99 linearity. The input-referred noise was 6.9 µVrms and the flicker noise was negligible. The total power consumption was under 55 µW. The complete system was assembled on a 20 mm × 20 mm platform that includes the potentiostat chip, the electrode terminals and an instrumentation amplifier for redox current buffering, once converted to a voltage by a series resistor. the chip dimensions were 1 mm × 0.5 mm and the other PCB components were off-chip resistors, capacitors and amplifiers for data acquisition. The system was successfully tested with ferricyanide, a stable electroactive compound, and validated with dopamine, a popular neurotransmitter.

Gold nanoparticles in ophthalmology

Many research projects are underway to improve the diagnosis and therapy in ophthalmology. Indeed, visual acuity deficits affect 285 million people worldwide and different strategies are being developed to strengthen patient care. One of these strategies is the use of gold nanoparticles (GNP) for their multiple properties and their ability to be used as both diagnosis and therapy tools. This review exhaustively details research developing GNPs for use in ophthalmology. The toxicity of GNPs and their distribution in the eye are described through in vitro and in vivo studies. All publications addressing the pharmacokinetics of GNPs administered in the eye are extensively reviewed. In addition, their use as biosensors or for imaging with optical coherence tomography is illustrated. The future of GNPs for ophthalmic therapy is also discussed. GNPs can be used to deliver genes or drugs through different administration routes. Their antiangiogenic and anti‐inflammatory properties are of great interest for different ocular pathologies. Finally, GNPs can be used to improve stereotactic radiosurgery, brachytherapy, and photothermal therapy because of their many properties.