Rafael G. Oliveira

Bidirectional Control of Sphingomyelinase Activity and Surface Topography in Lipid Monolayers

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.

Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence

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+) and divalent (Ca2+) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazing-incidence X-ray fluorescence. In the absence of Ca2+, a K+ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl2, Ca2+ ions almost completely displaced K+ 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.

The self-organization of lipids and proteins of myelin at the membrane interface. Molecular factors underlying the microheterogeneity of domain segregation.

The advances over the last 10 years on the understanding of myelin heterogeneity are reviewed. The main focus is on the applicability of Langmuir monolayers, Langmuir-Blodgett films and some associated techniques to unravelling the behaviour of interfaces formed with all the components of a natural membrane. Lipid-protein lateral segregation appears as a major driving force to determine surface patterns that can change under compression from circular domains to two-dimensional fractal structures. The major proteins of the myelin membrane induce lateral segregation in an otherwise homogeneous surface formed by the mixture of total myelin lipids. The lipid and protein components appear to distribute in the surface domains according to their charge, compressibility and relative molecular weight: myelin proteins, ganglioside GM1 and fluorescent lipid probes partition into liquid-expanded phase domains; other components such as phosphatidylserine and galactocerebroside partition into another liquid phase enriched in cholesterol. Simplified protein-lipid mixtures allow assessment of the participation of the major proteins in the two dimensional pattern development. One of the major myelin proteins, the Folch-Lees proteolipid, self-segregates into, and determines formation of, fractal-like patterns. The presence of the second major protein, myelin basic protein, leads to round liquid-expanded domains in the absence of Folch-Lees proteolipid and softens the boundaries of the fractal structures in its presence. The location of myelin basic protein in the interface is surface pressure sensitive, being slightly squeezed out at high surface pressure, allowing the fractal domains enriched in Folch-Lees proteolipid to evolve.

Surface behavior of myelin monolayers

Myelin can be spread as a stable monomolecular layer, with reproducible properties, at the air-water interface. The major lipids and proteins of myelin are represented in this monolayer in molar ratios similar to those in the original membrane. A well-defined collapse point of the myelin monolayer occurs at ca. 46 mN/m. At a surface pressure of ca. 20 mN/m, the surface pressure-molecular area isotherm of the myelin monolayer shows a change in its compressibility, exhibited as a diffuse but reproducible inflection with a clearly marked change of the surface compressional modulus; the surface potential-area curve shows a change of slope at the same surface pressure. The myelin monolayer shows considerable hysteresis during the first compression-decompression cycle; no detectable protein unfolding under expansion; and decreased hysteresis after the first cycle. The average molecular areas, the inflection at 20 mN/m, the variation of the surface potential per unit of molecular surface density, and the hysteresis properties of the myelin monolayer indicate that this membrane undergoes changes of intermolecular organization mostly ascribed to the protein fraction, above a lateral surface pressure of ca. 20 mN/m. The behavior is consistent with a surface pressure-dependent relocation of protein components in the film. This has marked effects on the stability, molecular packing, and dipolar organization of the myelin interface.

Composition-driven surface domain structuring mediated by sphingolipids and membrane-active proteins. Above the nano- but under the micro-scale: mesoscopic biochemical/structural cross-talk in biomembranes.

Biomembranes contain a wide variety of lipids and proteins within an essentially two-dimensional structure. The coexistence of such a large number of molecular species causes local tensions that frequently relax into a phase or compositional immiscibility along the lateral and transverse planes of the interface. As a consequence, a substantial microheterogeneity of the surface topography develops and that depends not only on the lipid–protein composition, but also on the lateral and transverse tensions generated as a consequence of molecular interactions. The presence of proteins, and immiscibility among lipids, constitute major perturbing factors for the membrane sculpturing both in terms of its surface topography and dynamics. In this work, we will summarize some recent evidences for the involvement of membrane-associated, both extrinsic and amphitropic, proteins as well as membrane-active phosphohydrolytic enzymes and sphingolipids in driving lateral segregation of phase domains thus determining long-range surface topography.

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

Epifluorescence Microscopy of Surface Domain Microheterogeneity in Myelin Monolayers at the Air-Water Interface

Myelin lipids form liquid-expanded monolayers at the air-water interface, with no evidence of surface pressure-induced two-dimensional phase transition. However, the film doped with 2 mole % of the fluorescent probe N-(7-nitro-2-1,3-benzoxadiazol-4-yl) Diacyl Phosphatidyl-ethanolamine (NBD-PE) shows an irregular pattern of coexisting laterally segregated surface domains with diffuse boundaries that change from smooth patterns to fractal-like structures depending on surface pressure. Successive expansion-recompression cycles lead to more defined domains, with a general reorganization occurring at surface pressures of about 20 mN/m. At least two coexisting phases occur over almost all the compression isotherms. The presence of proteins in whole myelin monolayers induces defined domain textures with relatively sharp boundaries. The patterns during compression and expansion are quite similar and, after the first cycle, little changes occur under recompression. The patterns observed provide topographical evidence for the existence of dynamic domain microheterogeneity in the surface of myelin interfaces.

Surface behavior, microheterogeneity and adsorption equilibrium of myelin at the air-water interface.

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.

Biochemical and structural information transduction at the mesoscopic level in biointerfaces containing sphingolipids

In this work we describe two aspects of molecular and supramolecular information transduction. The first is the biochemical and structural information content and transduction associated with sphingomyelinase activity. The results disclose a lipid-mediated cross-communication between the sphingomyelinase and phospholipase A2 pathways. In addition, the two-dimensional degradation of sphingomyelin by sphingomyelinase affects the surface topography and the latter modulates the enzyme activity. The second is the information contained in the compositionally driven lateral organization of whole glial and neuronal membrane interfaces. The myelin monolayer exhibits microheterogeneous topographical structuring and nonhomogeneous lateral thickness of phase separated regions, depending dynamically on the lateral surface pressure. On the other hand, the differential response of functional living cells depends on information contained in the molecular organization of the contacting membrane interface.

Refractive index and thickness determination in Langmuir monolayers of myelin lipids

Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.