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.

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.

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.

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.

Effect of surface charge on the interfacial orientation and conformation of FB1 in model membranes.

The toxicity of FB1 is usually explained through the enzymatic disruption of lipidic metabolism. However, it may lie in the thermodynamics of the membrane and its cooperative phase behavior rather than in the activity of individual proteins. Here, we investigate the effects of FB1 at the molecular and mesoscopic levels in FB1-phospholipid mixed Langmuir films. Mean molecular area vs FB1 molar fractions (x(FB1)) and phase diagram analysis allowed us to define miscibility conditions and phase states at different x(FB1). Surface potential measurements, evaluated as a function of the molecular packing and x(FB1), revealed the FB1-induced change in the collective dipolar reorientation leaded to neutralization of charged films. Size, shape, and distribution of 2D-domain analysis from epifluorescence data suggested the increase in the mixing entropy and film relaxation rate. Finally, PM-IRRAS revealed the orientation of FB1 with the amine end (zwitterionic and negatively charged monolayers) or the tricarballylic acid end (positively charged monolayers) pointing to the air. The globular-extended conformational equilibrium of FB1 is dynamically defined by the membrane charge becoming a toxicity enhancing factor. The specificity of the toxin-protein interaction might also be perturbed by the FB1-induced remodeling of the membrane topography by affecting the raft-like platforms where membrane enzymes are considered to be located.

Activity modulation and reusability of beta-D-galactosidase confined in sol-gel derived porous silicate glass.

In the present work we applied the sol-gel method to obtain glass lentils entrapping beta-D-galactosidase (beta-Gal) (Ebeta-Gal) within a silicate matrix. The effect of pH, temperature, polarity and salt concentration on the activity of Ebeta-Gal was studied. Apparent kinetic parameters for ortho-nitro-phenyl-beta-D-galactopyranoside hydrolysis catalyzed by Ebeta-Gal (V'max, K'M) were lower compared to the soluble enzyme (Sbeta-Gal), reflecting the solute diffusion restriction imposed by the matrix observed in the time curves, a partial protein inactivation upon encapsulation, and an improvement in the affinity of Ebeta-Gal for the substrate as compared with Sbeta-Gal. At pH<4, Ebeta-Gal stability was higher than that of Sbeta-Gal. Ebeta-Gal could be reused after storage at 4 degrees C for up to 90 days, and retained its activity profile within the range of pH=2-10 and saline concentration 0-400 mM. Pre-incubation at 75 degrees C for 30 min fully inactivated Sbeta-Gal while Ebeta-Gal retained approximately 90% of its activity, even in the reused samples. Encapsulation did not introduce additional impairments to the reaction rate measured in heterogeneous dispersions, beyond those derived from their own particle-crowded environment. This reusable Ebeta-Gal was resistant to typical technological conditions applied in milk processing that would lead to the unfolding and inactivation of Sbeta-Gal. The results are discussed from the biophysical viewpoint.

Molecular packing tunes the activity of Kluyveromyces lactis beta-galactosidase incorporated in Langmuir-Blodgett films.

Functional consequences of constraining beta-Gal in bidimensional space were studied at defined molecular packing densities and constant topology. Langmuir-Blodgett films, LB15 and LB35 composed of dipalmitoyl phosphatidylcholine and K. lactis beta-Gal, were obtained by transferring Langmuir films (L) initially packed at 15 and 35 mN/m, respectively, to alkylated glasses. The beta-Gal-monolayer binding equilibrium, mainly the adsorption rate and affinity, depended on the initial monolayer's surface pressure (lower for higher pi i). At pi i = 15 and 35 mN/m, the surface excess (Gamma) followed downward parabolic and power-law tendencies, respectively, as a function of subphase protein concentration. Gamma values in L roughly reflected the protein surface density chemically determined in LBs (0-7.5 ng/mm2 at pi i = 0-35 mN/m and [beta-Gal] subphase = 0-100 microg/mL). The beta-Gal-catalyzed hydrolysis of o-nitrophenyl-galactopyranoside showed a Michaelian kinetics in solution as well as in LB15. KM, KM,LB15, Vmax, and Vmax,LB15 were 5.15 +/- 2.2 and 9.25 +/- 6 mM and 39.63 and 0.0096 +/- 0.0027 micromol/min/mg protein, respectively. The sigmoidal kinetics observed with LB35 was evaluated by Hill's model (K0.5 = 9.55 +/- 0.4 mM, Vmax,35 = 0.0021 micromol/min/mg protein, Hill coefficient n = 9) and Savageau's fractal model (fractal constant K f = 9.84 mM; reaction order for the substrate gs = 9.06 and for the enzyme ge = 0.62). Fractal reaction orders would reflect the fractal organization of the environment, demonstrated by AFM images, more than the molecularity of the reaction. Particular dynamics of the protein-lipid structural coupling in each molecular packing condition would have led to the different kinetic responses.