Concepción Civera

Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl‐10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique α/β fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three‐dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix α4 inserted in the β4‐β5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix α4 may also be present in the DEP domains of regulator of G‐protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N‐terminal PH domain in pleckstrin. Phosphorylation of the PH–DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far. Proteins 2005.

Efficient and selective enzymatic synthesis of N-acetyl-lactosamine in ionic liquid: a rational explanation

Room temperature ionic liquids (ILs) can affect enzyme activity in some enzyme-catalyzed reactions, however the effects of these cosolvents on the enzymes are not clearly understood. Using β-galactosidase from Thermus thermophilus HB27 (TTP0042), we found an important change from the classical regioselectivity of the transglycosylation reaction with this enzyme. The enzyme increases N-acetyl-D-lactosamine synthesis (Galβ[1→4]GlcNAc) when RTILs are used instead of the traditional self-condensated products. To understand the possible effect of these liquids on the synthetic behavior of the enzyme, we performed a molecular interaction study by surface plasmon resonance. The KD value obtained for this interaction could mean that ILs bind to β-galactosidase through non specific interactions characterized by very fast kinetics and millimolar affinity. Then, several reactions were performed, increasing the concentration of the IL. As a result, a dependence on the ILs concentration was found for transglycosylation products. We hypothesize that ILs might induce conformational changes in the enzyme, which would modify the enzymatic activity and regioselectivity. These structural modifications were confirmed in the secondary and tertiary structures of the protein by circular dichroism and fluorescence studies, respectively. Molecular modeling confirms this hypothesis and shows that the enzyme becomes more flexible in an IL–water mixture and that it allows stabilization of the GlcNAc molecule in the active centre of the enzyme, in order to develop a new product according to the original regioselectivity of the reaction.

Interactions of 2,2,2-trifluoroethanol with aqueous micelles of Triton X-100

The effect of 2,2,2-trifluoroethanol (TFE) on micellar properties of Triton X-100 (TX-100) in aqueous solutions was investigated by cloud point (CP), viscosity, surface tension, and fluorescence techniques. The critical micelle concentration (CMC) values of the corresponding mixtures were obtained by the pyrene 1:3 ratio method and by surface tension data using the pendant drop technique. All the techniques provided about the same values for the CMC. Up to 0.83 M TFE increased the CMC by 30%. The small increase in the CMC is consistent with a slight increase in the solubility of the TX-100. Fluorescence measurements indicate that the TFE decreased the aggregation number by about 30%. The CP decrease and the intrinsic viscosity increase with TFE concentration are consistent with a preferential interaction of TFE with TX-100 micelles. TFE molecules form hydrophobic domains in the micellar layer palisade because they hydrogen bond with the oxyethylene group in TX-100. The intrinsic viscosity data are consistent with an increase in micelle hydrodynamic radius owing to the presence of TFE.

Highly efficient and regioselective enzymatic synthesis of β-(1→3) galactosides in biosolvents

A green synthesis for β-(1→3) galactosyldisaccharides that combines the use of a biodegradable biocatalyst, aqueous solutions, and solvent recycling (renewable and derived from biomass) has been developed. The use of biomass-derived solvents allows good catalytic activity in the synthesis of Gal-β-D-(1→3)GlcNAc and Gal-β-D-(1→)3GalNAc (99% and 95% yields respectively) with β-Gal-3-NTag β-galactosidase, preventing hydrolytic activity and with full regioselectivity. This represents a considerable improvement over the use of an aqueous buffer or conventional organic solvents. Furthermore, reaction scaling up and biosolvent recycling are feasible without losing catalytic action. In order to understand the role of these green solvents in the enzymes synthetic behaviour, different structural studies were performed (fluorescence and molecular modelling) in the presence of some selected biosolvents to conclude that the presence of green biosolvents in the reaction media modifies the enzymes tertiary structure allowing better substrate disposition in the active site, most probably due to solvation effects, explaining the behaviour observed.

Cosmetics and Cosmeceutical Applications of Chitin, Chitosan and Their Derivatives

Marine resources are well recognized for their biologically active substances with great potential applications in the cosmeceutical industry. Among the different compounds with a marine origin, chitin and its deacetylated derivative—chitosan—are of great interest to the cosmeceutical industry due to their unique biological and technological properties. In this review, we explore the different functional roles of chitosan as a skin care and hair care ingredient, as an oral hygiene agent and as a carrier for active compounds, among others. The importance of the physico-chemical properties of the polymer in its use in cosmetics are particularly highlighted. Moreover, we analyse the market perspectives of this polymer and the presence in the market of chitosan-based products.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.