A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K þ ) and divalent (Ca 2þ ) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazingincidence X-ray fluorescence. In the absence of Ca 2þ , a K þ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl 2 , Ca 2þ ions almost completely displaced K þ ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.monolayer | Monte Carlo simulation | electrostatics | biological interface B iological surfaces expose a variety of charged macromolecules that interact with various sorts of ions under physiological conditions. However, despite the crucial role of charged macromolecules in modulating the interaction between cells and their surrounding environments, the quantitative understanding of electrostatics at such soft, complex interfaces still remains a general scientific challenge. For example, the outer membrane surface of Gram-negative bacteria is mainly composed of lipopolysaccharides (LPSs) (1), whose negatively charged saccharide head groups stabilize the structural integrity of bacteria and protect bacteria against their environment. Several in vivo studies (2, 3) demonstrated that bacteria increase their resistance against cationic antimicrobial peptides (e.g., protamine) in the presence of divalent cations (Ca 2þ , Mg 2þ ). Therefore, for the development of peptide-based antibiotics (4), it is important to understand the molecular mode of action of antimicrobial peptides.A number of theoretical models for the interactions of LPS molecules with divalent cations (5-7), suggested that the ions would bind to the charged 2-keto-3-deoxyoctonoic acid (KDO) groups (the "inner core") thereby stabilizing the membrane. Recently, we measured grazing-incidence X-ray scattering from a monolayer of rough mutant LPS from Salmonella enterica sv. Minnesota at an air/water interface and demonstrated the Ca 2þ -induced increase in the electron density near the inner core (8). These observations were supported by the results of Monte Carlo (MC) simulations of a coarse-grained model (8). A further challenge would be to extend such a strategy to wild-type LPSs that possess polydisperse, specific O-polysaccharide chains (O-side chains). Pink et al. (9) carried out MC simulations of a minimal model of the more complex, wild-type LPSs from Pseudomonas aeruginosa (PAO1) and concluded that divalent cations would induce a collapse of the negatively charged O-sidechains toward the membrane surface. Because it is difficult in practice to deposit LPSs wit...
Lipid lateral organization is increasingly found to modulate membrane-bound enzymes. We followed in real time the reaction course of sphingomyelin (SM) degradation by Bacillus cereus sphingomyelinase (SMase) of lipid monolayers by epifluorescence microscopy. There is evidence that formation of ceramide (Cer), a lipid second messenger, drives structural reorganization of membrane lipids. Our results provide visual evidence that SMase activity initially alters surface topography by inducing phase separation into condensed (Cer-enriched) and expanded (SM-enriched) domains. The Cer-enriched phase grows steadily as the reaction proceeds at a constant rate. The surface topography derived from the SMase-driven reaction was compared with, and found to differ from, that of premixed SM/Cer monolayers of the same lipid composition, indicating that substantial information content is stored depending on the manner in which the surface was generated. The long-range topographic changes feed back on the kinetics of Smase, and the onset of condensed-phase percolation is temporally correlated with a rapid drop of reaction rate. These observations reveal a bidirectional influence and communication between effects taking place at the local molecular level and the supramolecular organization. The results suggest a novel biocatalytic-topographic mechanism in which a surface enzymatic activity can influence the function of amphitropic proteins important for cell function.
The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.
The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.
Grazing incidence x-ray scattering techniques and Monte Carlo (MC) simulations are combined to reveal the influence of molecular structure (genetic mutation) and divalent cations on the survival of gram negative bacteria against cationic peptides such as protamine. The former yields detailed structures of bacterial lipopolysaccharide (LPS) membranes with minimized radiation damages, while the minimal computer model based on the linearized Poisson-Boltzmann theory allows for the simulation of conformational changes of macromolecules (LPSs and peptides) that occur in the time scale of ms. The complementary combination of the structural characterizations and MC simulation demonstrates that the condensations of divalent ions (Ca2+ or Mg2+) in the negatively charged core saccharides are crucial for bacterial survival.
In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase)-driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the mum level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.
Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.
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