The role of water in reactions catalysed by hydrolases under conditions of molecular crowding.

Under macromolecular crowding (MC) conditions such as cellular, extracellular, food and other environments of biotechnological interest, the thermodynamic activity of the different macromolecules present in the system is several orders of magnitude higher than in dilute solutions. In this state, the diffusion rates are affected by the volume exclusion induced by the crowders. Immiscible liquid phases, which may arise in MC by liquid-liquid phase separation, may induce a dynamic confinement of reactants, products and/or enzymes, tuning reaction rates. In cellular environments and other crowding conditions, membranes and macromolecules provide, on the whole, large surfaces that can perturb the solvent, causing its immobilisation by adsorption in the short range and also affecting the solvent viscosity in the long range. The latter phenomenon can affect the conformation of a protein and/or the degree of association of its protomers and, consequently, its activity. Changes in the water structure can also alter the enzyme-substrate interaction, and, in the case of hydrolytic enzymes, where water is one of the substrates, it also affects the reaction mechanism. Here, we review the evidence for how macromolecular crowding affects the catalysis induced by hydrolytic enzymes, focusing on the structure and dynamics of water.

Nanoarchitectonic approaches for measuring the catalytic behavior of a membrane anchored enzyme. From Langmuir-Blodgett to a novel Langmuir-Schaefer based nanofilm building device.

Self-organized lipid monolayers at the air-water interface (Langmuir films, LF) are commonly used for measuring the catalytic properties of membrane-bound enzymes. This methodology allows to provide a consistent flat topography molecular density, packing defects and thickness. The aim of the present work was to show the methodological advantages of using the horizontal transfer method (Langmuir-Schaefer) with respect to the vertical transfer method (Langmuir-Blodgett) when mounting a device to measure catalytic activity of membrane enzymes. Based on the results obtained we can conclude that it is possible to prepare stable Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) films from Bovine Erythrocyte Membranes (BEM) preserving the catalytic activity of its native Acetylcholinesterase (BEA). In comparison, the LS films showed Vmax values more similar to the enzyme present in the vesicles of natural membranes. In addition, it was much easier to produce large amounts of transferred areas with the horizontal transfer methodology. It was possible to decrease the time required to mount an assay with numerous activity points, such as building activity curves as a function of substrate concentration. The present results show that LSBEM provides a proof of concept for the development of biosensors based on transferred purified membrane for the screening of new products acting on an enzyme embedded on its natural milieu. In the case of BEA, the application of these enzymatic sensors could have medical interest, providing drug screening tools for the treatment of Alzheimer's disease.

His-tag ß-galactosidase supramolecular performance.

ß-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant ß-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (ß-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (ß-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of ß-GalHis with respect to ß-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, ß-GalHis presented a higher resistance to the thermal inactivation compared to ß-GalWT. At room temperature, ß-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to ß-GalWT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability.

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of ß-Galactosidase from Kluyveromyces lactis.

Here, we report the effect of polyethylene glycol (PEG6000)-induced molecular crowding (MC) on the catalytic activity and thermal stability of Kluyveromyces lactis ß-galactosidase (ß-Gal). The ß-Gal-catalyzed hydrolysis of o-nitrophenyl-ß-d-galactopyranoside followed a Michaelian kinetics at [PEG6000] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG6000). Compared with dilute solutions, in the MC media, ß-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (e.g., 45 °C) and by the slower inactivation kinetics. Considering the effects of water thermodynamic activity on the reaction kinetics and protein structure and the effect of the exclusion volume on protein conformation, we suggest that changes in the protein oligomerization state and hydration could be the responsible for the behavior observed at the highest MC levels assayed. These results could be relevant and should be taken into account in industrial food processes applying ß-Gal from K. lactis.

Probing Thermotropic Phase Behavior of Dipalmitoylphosphatidylcholine Bilayers from Electrical and Topographic Data in a Horizontal Black Lipid Membrane Model.

Here, a homemade device allowed preparing horizontal lipid bilayer membranes (hBLMs) for recording electrical and topographical data simultaneously and in real-time, under temperature (T)-controlled conditions along a cooling process of dipalmitoylphosphatidylcholine (DPPC) bilayers. Electrical parameters (ionic current intensity, I, and transmembrane resistance, R = ΔV/I) plotted against T exhibited discontinuities at the main transition (TPß'→Lα) and pretransition (TLß→Pß') temperatures of DPPC. The T-dependent sensitivity to ΔV-induced electrostriction was revealed by capacitance measurements. The patterns of I fluctuation described long-range correlations reflected by 1/f-type noise in the ripple phase (Pß') and Brownian-type fluctuations in the liquid-crystalline (Lα) phase at voltage intensities lower than a voltage threshold (ΔVth = ±160 mV), indicating that autocorrelations arise from an underlying structural connectivity that takes place within ordered phases. At |V| ≥ Vth, the fluctuation dynamics exhibited a 1/f behavior over the whole temperature range analyzed, suggesting that upon a certain intensity of external electrical perturbation, the membrane system evolves toward a voltage-induced percolated-pore state. At T > TPß'→Lα, differential interference contrast micrographs showed droplet-like structures, probably containing solvent traces of the lipid solution, which were reverted upon cooling. However, droplets did not interfere with the thermotropic equilibrium of the bilayer phase. This suggested that the temperature-induced changes in the electrical properties of the bilayer, as well as in the complexity of the fluctuation patterns (emergency of long- and short-range correlations), were strongly associated with the characteristic thermotropic behavior of DPPC, without significant deviations induced by the presence of residual n-decane in the bilayer. Our hBLM model membrane proved useful for correlating thermotropic phase changes with electro-biophysical and topographical information.

Sensing molecular organizational changes through the catalytic activity of acetylcholinesterase from erythrocyte membranes in Langmuir-Blodgett films.

Langmuir films prepared from bovine erythrocyte membranes (LFBEM) were studied and transferred to alkylated glasses (Langmuir-Blodgett films, LBBEM) in order to assess the effects of membrane molecular packing on Bovine Erythrocyte Acetylcholinesterase (BEA) catalytic activity. Surface pressure (π) vs Area isotherms showed three 2D-transitions at ~7, ~18 and ~44 mN/m and a collapse pressure at πc = 49 mN/m. The 0-12-0 mN/m compression-decompression cycles resulted reversible while those 0-40-0 mN/m exhibited a significant hysteresis. Taken together, EFM, BAM and AFM images and the stability of the film after 3C-D cycles, we can suggest that over the air-water interface as well as over the silanized glass substrate the surface is mostly covered by a monolayer with a few particles dispersed. Acetylthiocholine hydrolysis was assayed with BEA in bovine erythrocyte membrane suspensions (SBEM) and in LBBEM packed at 10 (LBBEM,10) and 35 mN/m (LBBEM,35), which gave the following kinetic parameters: Vmax = 3.41 ± 0.15, 0.021 ± 0.002 and 0.030 ± 0.003 nmol.min-1·µg prot-1 and KM = 0.11 ± 0.02, 0.047 ± 0.017 and 0.026 ± 0.017 mM, respectively. Although from SBEM to LBBEM we lost active enzyme, the catalytic efficiency (Vmax/KM) increased ~750 times. Eugenol and 1,8-cineol inhibited BEA catalytic activity in LBBEM,35. Our results demonstrate the transmission of information between the membrane and the environment within the subphase immediately below the membrane, where anchored proteins are hosted. This was reflected by the membrane packing-induced modulation of BEA catalytic activity. Furthermore, LBBEM provides a proof of concept for the development of biosensors to screen new green pesticides acting through BEA interaction.

Dual substrate/solvent- roles of water and mixed reaction-diffusion control of ß-Galactosidase catalyzed reactions in PEG-induced macromolecular crowding conditions.

Here we studied the effect of molecular crowding on the hydrolysis of ortho- and para-nitrophenyl-ß-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli ß-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG6000). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG6000] as well as for ONPG in the absence of PEG6000 so a Michaelian model was applied to calculate the kinetic parameters KM and kcat (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG6000, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs. [ONPG] plots hence calculating two apparent KM and two kcat values. Since the rate limiting-step of the enzymatic hydrolysis mechanism of ONPG, but not of PNPG, is the water-dependent one, the existence of several molecular water populations differing in their energy and/or their availability as reactants may explain the biphasic kinetics in the presence of PEG6000. With PNPG, KM as well as kcat varied with [PEG6000] like a parabola opening upward with a minimum at 15 %w/v [PEG6000]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in kcat plus high and [PEG6000]-dependent increasing KM values. Sedimentation velocity analysis demonstrated that PEG6000 impaired the diffusion of ß-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG6000-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion.

Langmuir films at the oil/water interface revisited.

We studied monomolecular layers at the oil/water interface (O/Wint) in a Langmuir interfacial trough using egg-yolk phosphatidylcholine (EPC) (the model phospholipid) and Vaseline (VAS) as oil phase. The temporal dynamics in the surface pressure (π) evolution depended on the method (spreading/adsorption) used for monolayers preparation and reflected the different distribution of EPC between all the system compartments (bulk phases and interfaces). We distinguished between EPC located either stable at the interface or hopping between the interface and bulk phases. The size order of the apparent mean molecular area, at constant π, of EPC at different interfaces (EPCO/W > EPC/VAS0.02;A/W > EPCA/W), suggested that VAS molecules intercalated between the hydrocarbon chains of EPCO/W, at a molar fraction xVAS > 0.02. However, EPC/VAS0.02;A/W showed the highest compressional free energy. This leaded us to study the EPC/VAS0.02 mixture at A/W by Brewster Angle Microscopy (BAM), finding that upon compression VAS segregated over the monolayer, forming non-coalescent lenses (as predicted by the spreading coefficient S = -13 mN/m) that remained after decompression and whose height changed (increase/decrease) accompanied the compression/decompression cycle. At the O/Wint, while some VAS molecules remained at the interface up to the collapse, others squeezed out towards the VAS bulk phase with an energy requirement lower than towards the air.

ß-sheet to α-helix conversion and thermal stability of ß-Galactosidase encapsulated in a nanoporous silica gel.

The effect on protein conformation and thermal stability was studied for ß-Galactosidase (ß-Gal) encapsulated in the nanopores of a silicate matrix (Eß-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (Sß-Gal), Eß-Gal exhibited a higher content of α-helix structure. Heating Eß-Gal up to 75 °C caused a decrease in the content of ß-sheet structure and additional augments on Eß-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with Sß-Gal. Steady state fluorescence spectroscopy analysis evidenced an Eß-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for Sß-Gal the denaturation midpoint (Tm) was 59 °C, for Eß-Galit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and Eß-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of Eß-Gal leading to the loss of thermal stability and enzymatic activity.

Superactive ß-galactosidase inclusion bodies.

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant ß-galactosidase inclusion bodies (IBß-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBß-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis-Menten constant, measured in the IBß-Gal suspensions. Simultaneously, IBß-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBß-Gal represents a reservoir and packed source of highly active and stable enzyme.

Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity GABAA-R ligands and potential anxiolytics.

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [3H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABAA-R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5-1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test.

V-Shaped Molecular Configuration of Wax Esters of Jojoba Oil in a Langmuir Film Model.

The aim of the present work was to understand the interfacial properties of a complex mixture of wax esters (WEs) obtained from Jojoba oil (JO). Previously, on the basis of molecular area measurements, a hairpin structure was proposed as the hypothetical configuration of WEs, allowing their organization as compressible monolayers at the air-water interface. In the present work, we contributed with further experimental evidence by combining surface pressure (π), surface potential (Δ V), and PM-IRRAS measurements of JO monolayers and molecular dynamic simulations (MD) on a modified JO model. WEs were self-assembled in Langmuir films. Compression isotherms exhibited πlift-off at 100 Å2/molecule mean molecular area ( Alift-off) and a collapse point at πc ≈ 2.2 mN/m and Ac ≈ 77 Å2/molecule. The Δ V profile reflected two dipolar reorganizations, with one of them at A > Alift-off due to the release of loosely bound water molecules and another one at Ac < A < Alift-off possibly due to reorientations of a more tightly bound water population. This was consistent with the maximal SP value that was calculated according to a model that considered two populations of oriented water and was very close to the experimental value. The orientation of the ester group that was assumed in that calculation was coherent with the PM-IRRAS behavior of the carbonyl group with the C═O oriented toward the water and the C-O oriented parallel to the surface and was in accordance with their orientational angles (∼45 and ∼90°, respectively) determined by MD simulations. Taken together, the present results confirm a V shape rather than a hairpin configuration of WEs at the air-water interface.

Environmental Topology and Water Availability Modulates the Catalytic Activity of ß-Galactosidase Entrapped in a Nanosporous Silicate Matrix.

In the present work we studied the catalytic activity of E. coli ß-Gal confined in a nanoporous silicate matrix (Eß-Gal) at different times after the beginning of the sol-gel polymerization process. Enzyme kinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps of the reaction mechanism for their ß-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through 1H-NMR, and scanning electron microscopy analysis of the gel nanostructure, were performed. In conjunction, results provided evidence that water availability is crucial for the modulation observed in the catalytic activity of ß-Gal as long as water participate in the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrate concentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (kcB). More structured water populations (lower T2) correlates with higher catalytic rate constants (kcB). The T2-kcB negative correlation observed along the aging of gels within the 15-days period assayed reinforces the coupling between water structure and the hydrolysis catalysis inside gels.

Langmuir films of dipalmitoyl phosphatidylethanolamine grafted poly(ethylene glycol). In-situ evidence of surface aggregation at the air-water interface.

The molecular packing-dependent interfacial organization of polyethylene glycol grafted dipalmitoylphosphatidylethanolamine (PE-PEGs) Langmuir films was studied. The PEG chains covered a wide molecular mass range (350, 1000 and 5000Da). In surface pressure-area (π-A), isotherms PE-PEG1000 and PE-PEG5000 showed transitions (midpoints at πm,t1∼11mN/m, "t1"), which appeared as a long non-horizontal line region. Thus, t1 cannot be considered a first-order phase transition but may reflect a transition within the polymer, comprising its desorption from the air-water interface and compaction upon compression. This is supported by the increase in the νs(C-O-C) PM-IRRAS signal intensity and the increasing surface potentials at maximal compression, which reflect thicker polymeric layers. Furthermore, changes in hydrocarbon chain (HC) packing and tilt with respect to the surface led to reorientation in the PO2- group upon compression, indicated by the inversion of the νasym(PO2-) PM-IRRAS signal around t1. The absence of a t1 in PE-PEG350 supports the requisite of a critical polymer chain length for this transition to occur. In-situ epifluorescence microscopy revealed 2D-domain-like structures in PE-PEG1000 and PE-PEG5000 around t1, possibly associated with gelation/dehydration of the polymeric layer and appearing at decreasing π as the polymeric tail became longer. Another transition, t2, appearing in PE-PEG350 and PE-PEG1000 at πm,t2=29.4 and 34.8mN/m, respectively, was associated with HC condensation and was impaired in PE-PEG5000 due to steric hindrance imposed by the large size of its polymer moiety. Two critical lengths of polymer chains were found, one of which allowed the onset of polymeric-tail gelation and the other limited HC compaction.

Improved prediction of bilayer and monolayer properties using a refined BMW-MARTINI force field.

Coarse-grained (CG) models allow enlarging the size and time scales that are reachable by atomistic molecular dynamics simulations. A CG force field (FF) for lipids and amino acids that possesses a polarizable water model has been developed following the MARTINI parametrization strategy, the BMW-MARTINI [1]. We tested the BMW-MARTINI FF capability to describe some structural and thermodynamical properties of lipid monolayers and bilayers. We found that, since the surface tension values of oil/water interfaces calculated with the model are not correct, compression isotherms of lipid monolayers present artifacts. Also, this FF predicts DPPC and DAPC bilayers to remain in the Lα phase at temperatures as low as 283K, contrary to the expected from their experimental Tm values. Finally, simulations at constant temperature of bilayers of saturated lipids belonging to PC homologous, showed an increase in the mean molecular area (Mma) upon increasing the chain length, inversely to the experimental observation. We refined BMW-MARTINI FF by modifying as few parameters as possible in order to bring simulated and experimental measurements closer. We have also modified structural parameters of the lipid geometry that do not have direct influence in global properties of the bilayer membranes or monolayers, but serve to approach the obtained CG geometry to atomistic reference values. The refined FF is able to better reproduce phase transition temperatures and Mma for saturated PC bilayers than BMW-MARTINI and MARTINI FF. Finally, the simulated surface pressure-Mma isotherms of PC monolayers resemble the experimental ones and eliminate serious artifacts of previous models.

PEG-induced molecular crowding leads to a relaxed conformation, higher thermal stability and lower catalytic efficiency of Escherichia coli ß-galactosidase.

Enzymatic activities were historically assayed in dilute solutions where molecular crowding, molecular confinement and their consequences were not taken into account. Here we report how macromolecular crowding tunes catalytic parameters for the tetrameric ß-Galactosidase from Escherichia coli, ß-Gal. We detected increases in KM (weaker substrate binding) and a nonlinear variation in Vmax, with a minimum at 25% W/P of the crowding agent (polyethyleneglycol molecular mass 6000, PEG(6000)) resulting in a linear decrease in the catalytic efficiency (kcat/KM) within the whole [PEG(6000)] range tested). Presence of crowding agent affected ß-Gal structural content and increased its thermal resistance. Steady state fluorescence and Fourier transformed infrared spectroscopic observations are compatible with crowding-induced disordering and restricted internal dynamics as a result of excluded volume and solvent structuring effects. This leads to a non-optimal substrate-binding site and a less conformationally strained protein.

Synaptosomal membrane-based Langmuir-Blodgett films: a platform for studies on γ-aminobutyric acid type A receptor binding properties.

In this work we used Langmuir-Blodgett films (LB) as model membranes to study the effect of molecular packing on the flunitrazepam (FNZ) accessibility to the binding sites at the GABAA receptor (GABAA-R). Ligand binding data were correlated with film topography analysis by atomic force microscopy images (AFM) and SDS-PAGE. Langmuir films (LF) were prepared by the spreading of synaptosomal membranes (SM) from bovine brain cortex at the air-water interface. LBs were obtained by the transference, at 15 or 35 mN/m constant surface pressure (π), of one (LB15/1c and LB35/1c) or two (LB35/2c) LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference was performed in a serial manner, which allowed the accumulation of a great number of samples. SDS-PAGE clearly showed a 55 kDa band characteristic of GABAA-R subunits. Detrended fluctuation analysis of topographic data from AFM images exhibited a single slope value (self-similarity parameter α) in CONglass and a discontinuous slope change in the α value at an autocorrelation length of ∼100 nm in all LB samples, supporting the LF transference to the substrate. AFM images of CONglass and LB15/1c exhibited roughness and average heights that were similar between measurements and significantly lower than those of LB35/1c and LB35/2c, suggesting that the substrate coverage in the latter was more stable than in LB15/1c. While [(3)H]FNZ binding in LB15/1c did not reach saturation, in LB35/1c the binding kinetics became sigmoid with a binding affinity lower than in the SM suspension. Our results highlight the π dependence of both binding and topological data and call to mind the receptor mechanosensitivity. Thus, LB films provide a tool for bionanosensing GABAA-R ligand binding as well as GABAA-R activity modulation induced by the environmental supramolecular organization.

Water and membrane dynamics in suspensions of lipid vesicles functionalized with poly(ethylene glycol)s.

The present work was aimed at studying the molecular dynamics at different levels of model membranes having a simulated glycoclix, with focus on the molecular crowding conditions at the lipid-water interfacial region. Thus, binary mixtures of dipalmitoylphosphatidylcholine (dpPC) and a poly(ethylene glycol) (PEG(n)) derivative of dipalmitoylphosphatidylethanolamine (PE) (where n = 350, 1000, and 5000, respectively, refer to PEG molecular masses) were submitted to (1)H spin-lattice relaxation time (T1) and (31)P NMR spectra analysis. (1)H NMR relaxation times revealed two contributing components in each proton system (PEG, phospholipids, and water), for all the mixtures studied, exhibiting values of T1 with very different orders of magnitude. This allowed identifying confined and bulk water populations as well as PEG moieties becoming more disordered and independent from the phospholipid moiety as n increased. (31)P spectra showed a typical broad bilayer signal for n = 350 and 1000, and an isotropic signal characteristic of micelles for n = 5000. Surface pressure (π)-molecular area isotherms and compressional modulus measurements provided further structural information. Moreover, epifluorescence microscopy data from monolayers at π ∼ 30 mN/m, the expected equilibrium π in lipid bilayers, allowed us to postulate that both (1)H populations resolved through NMR in phospholipids and lipopolymers corresponded to different phase domains.

Cholesterol favors the emergence of a long-range autocorrelated fluctuation pattern in voltage-induced ionic currents through lipid bilayers.

The present paper was aimed at evaluating the effect of cholesterol (CHO) on the voltage-induced lipid pore formation in bilayer membranes through a global characterization of the temporal dynamics of the fluctuation pattern of ion currents. The bilayer model used was black lipid membranes (BLMs) of palmitoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine (POPE:POPC) at a 7:3 molar ratio in the absence (BLM0) or in the presence of 30 (BLM30), 40 (BLM40) or 50(BLM50)mol% of cholesterol with respect to total phospholipids. Electrical current intensities (I) were measured in voltage (ΔV) clamped conditions at ΔV ranging between 0 and ±200mV. The autocorrelation parameter α derived from detrended fluctuation analysis (DFA) on temporal fluctuation patterns of electrical currents allowed discriminating between non-correlated (α=0.5, white noise) and long-range correlated (0.5<α<1) behaviors. The increase in |ΔV| as well as in cholesterol content increased the number of conductance states, the magnitude of conductance level, the capacitance of the bilayers and increased the tendency towards the development of long-range autocorrelated (fractal) processes (0.5<α<1) in lipid channel generation. Experiments were performed above the phase transition temperature of the lipid mixtures, but compositions used predicted a superlattice-like organization. This leads to the conclusion that structural defects other than phase coexistence may promote lipid channel formation under voltage clamped conditions. Furthermore, cholesterol controls the voltage threshold that allows the percolation of channel behavior where isolated channels become an interconnected network.

ß-galactosidase at the membrane-water interface: a case of an active enzyme with non-native conformation.

Previously we demonstrated that Escherichia coli beta-galactosidase (ß-Gal) binds to zwitterionic lipid membranes improving its catalytic activity. To understand the activation mechanism from the protein perspective, here the thermal dependence of the catalytic activity was evaluated in conjunction with parameters derived from spectroscopy and calorimetry, in the presence and absence of egg-yolk phosphatidylcholine vesicles. In solution, the native state of ß-Gal exhibits a loose conformation according to the λmax of fluorescence emission, which is in the upper end of the emission range for most proteins. A non-two state thermal unfolding mechanism was derived from DSC experiments and supported by the sequential unfolding temperatures exhibited by fluorescence (55°C) and CD (60°C) spectroscopies. Quenching of ß-Gal's intrinsic fluorescence, provided evidence for a novel and even looser folding for the lipid-bound protein. However, DSC data showed that the thermal unfolding in the presence of lipids occurred with a significant decrease in ΔH compared to what happened in solution, suggesting that only the population of non-bound protein molecules were involved in this process. Concluding, upon binding to a lipid-water interface ß-Gal becomes trapped in a partially unfolded state, more active than that of the native protein in solution.