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.

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies.

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions.

His-tag β-galactosidase supramolecular performance.

β-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli).

Biofabrication of functional protein nanoparticles through simple His-tag engineering.

We have developed a simple, robust, and fully transversal approach for the fabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Zn ions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks.

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

Here, we report the effect of polyethylene glycol (PEG)-induced molecular crowding (MC) on the catalytic activity and thermal stability of β-galactosidase (β-Gal). The β-Gal-catalyzed hydrolysis of -nitrophenyl-β-d-galactopyranoside followed a Michaelian kinetics at [PEG] ≤ 25% w/v and positive cooperativity at higher concentrations (35% w/v PEG). Compared with dilute solutions, in the MC media, β-Gal exhibited stronger thermal stability, as shown by the increase in the residual activity recovered after preincubation at high temperatures (, 45 °C) and by the slower inactivation kinetics.

Artificial Inclusion Bodies for Clinical Development.

Bacterial inclusion bodies (IBs) are mechanically stable protein particles in the microscale, which behave as robust, slow-protein-releasing amyloids. Upon exposure to cultured cells or upon subcutaneous or intratumor injection, these protein materials secrete functional IB polypeptides, functionally mimicking the endocrine release of peptide hormones from secretory amyloid granules. Being appealing as delivery systems for prolonged protein drug release, the development of IBs toward clinical applications is, however, severely constrained by their bacterial origin and by the undefined and protein-to-protein, batch-to-batch variable composition.

Dual substrate/solvent- roles of water and mixed reaction-diffusion control of β-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-β-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli β-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG] as well as for ONPG in the absence of PEG so a Michaelian model was applied to calculate the kinetic parameters K and k (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs.

Superactive β-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 β-galactosidase inclusion bodies (IB) are functional aggregates.

PEG-induced molecular crowding leads to a relaxed conformation, higher thermal stability and lower catalytic efficiency of Escherichia coli β-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 β-Galactosidase from Escherichia coli, β-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).

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

Previously we demonstrated that Escherichia coli beta-galactosidase (β-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 β-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.

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.

Kinetics of lipid-membrane binding and conformational change of L-BABP.

We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group.

Chicken liver bile acid-binding protein is in a compact partly folded state at acidic pH. Its relevance to the interaction with lipid membranes.

Chicken liver bile acid-binding protein (formerly known as chicken liver basic fatty acid-binding protein) binds to anionic lipid membranes acquiring a partly folded state [Nolan, V., Perduca, M., Monaco, H.

Surface activity of thymol: implications for an eventual pharmacological activity.

In the present work, we studied the ability of thymol to affect the organization of model membranes and the activity of an intrinsic membrane protein, the GABA(A) receptor (GABA(A)-R). In this last aspect, we tried to elucidate if the action mechanism of this terpene at the molecular level, involves its binding to the receptor protein, changes in the organization of the receptor molecular environment, or both. The self-aggregation of thymol in water with a critical micellar concentration approximately = 4 microM and its ability to penetrate in monomolecular layers of soybean phosphatidylcholine (sPC) at the air-water interface, even at surface pressures above the equilibrium, lateral pressure of natural bilayers were demonstrated.

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes.

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air-water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl.