Lipid Phases and Cell Geometry During the Cell Cycle of Streptococcus pneumoniae.

The coexistence of different lipid phases is well-known in vitro, but evidence for their presence and function in cellular membranes remains scarce. Using a combination of fluorescent lipid probes, we observe segregation of domains that suggests the coexistence of liquid and gel phases in the membrane of Streptococcus pneumoniae, where they are localized to minimize bending stress in the ellipsoid geometry defined by the cell wall. Gel phase lipids with high bending rigidity would be spontaneously organized at the equator where curvature is minimal, thus marking the future division site, while liquid phase membrane maps onto the oblong hemispheres. In addition, the membrane-bound cell wall precursor with its particular dynamic acyl chain localizes at the division site where the membrane is highly curved. We propose a complete "chicken-and-egg" model where cell geometry determines the localization of lipid phases that positions the cell division machinery, which in turn alters the localization of lamellar phases by assembling the cell wall with a specific geometry.

Substitutions in PBP2b from ß-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity.

Pneumococcus resists ß-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because ß-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different ß-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism.

Phosphatidylserine Allows Observation of the Calcium-Myristoyl Switch of Recoverin and Its Preferential Binding.

Recoverin undergoes a calcium-myristoyl switch during visual phototransduction. Indeed, calcium binding by recoverin results in the extrusion of its myristoyl group, which allows its membrane binding. However, the contribution of particular lipids and of specific amino acids of recoverin in its membrane binding has not yet been demonstrated. In the present work, the affinity of recoverin for the negatively charged phosphatidylserine has been clearly shown to be governed by a cluster of positively charged residues located in its N-terminal segment. Moreover, the calcium-myristoyl switch of recoverin was only observed upon binding onto monolayers of phosphatidylserine and not in the case of other anionic phospholipids. Fluorescence microscopy experiments with mixed lipid monolayers allowed confirmation of the specific binding of myristoylated recoverin to phosphatidylserine, whereas the extent of penetration of recoverin in phosphatidylserine monolayers was estimated by ellipsometry. A model has thus been proposed for the membrane binding of myristoylated recoverin in the presence of calcium.

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 ß-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.

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.

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/ß domain of RP2 strongly suggest that the ß helix of RP2 plays a major role in the preferential binding of this protein to phospholipids in the LC state.

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.

Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers.

The lipid monolayer model membrane is useful for studying the parameters responsible for protein and peptide membrane binding. Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. This review focuses on the use of the "maximum insertion pressure" (MIP) to estimate the extent of protein and peptide penetration in lipid monolayers. The MIP data obtained with different proteins and peptides have been reviewed and discussed which allowed to draw conclusions on the parameters modulating the monolayer binding of proteins and peptides. In particular, secondary structure components such as amphipathic alpha-helices of proteins and peptides as well as electrostatic interactions play important roles in monolayer binding. The MIPs have been compared to the estimated lateral pressure of biomembranes which allowed to evaluate the possible association between proteins or peptides with natural membranes. For example, the MIP of a membrane-anchored protein with a glycosylphosphatidylinositol (GPI) was found to be far below the estimated lateral pressure of biomembranes. This allowed us to conclude that this protein is probably unable to penetrate the membrane and should thus be hanged at the membrane surface by use of its GPI lipid anchor. Moreover, the values of MIP obtained with myristoylated and non-myristoylated forms of calcineurin suggest that the myristoyl group does not contribute to monolayer binding. However, the acylation of a peptide resulted in a large increase of MIP. Finally, the physical state of lipid monolayers can have a strong effect on the values of MIP such that it is preferable to perform measurements with lipids showing a single physical state. Altogether the data show that the measurement of the maximum insertion pressure provides very useful information on the membrane binding properties of proteins and peptides although uncertainties must be provided to make sure the observed differences are significant.