Abel Moreno

Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

High resolution X-ray crystallographic structure of bovine heart cytochrome c and its application to the design of an electron transfer biosensor.

Cytochrome c is one of the most studied proteins probably due to its electron‐transfer properties in aerobic and anaerobic respiration. Particularly, cytochrome c from bovine heart is a small protein, Mr 12,230 Da, globular (hydrodynamic diameter of 3.4 nm), soluble in different buffer solutions, and commercially available. Despite being a quite well‐studied protein and relatively easy to manipulate from the biochemical and electrochemical viewpoint, its 3D structure has never been published. In this work, the purification, crystallization and 3D structure of one of the cytochrome c isoforms is presented to 1.5 Å resolution. It is also shown how the presence of isoforms made both the purification and crystallization steps difficult. Finally, a new approach for protein electrocrystallization and design of biosensors is presented. Proteins 2008.

The influence of an internal electric field upon protein crystallization using the gel-acupuncture method

In this work, the influence of an internal electric field upon the crystallization of lysozyme and thaumatin is explored using a modified design of the gel-acupuncture setup. From a crystallographic point of view, the orientation of crystals that grow preferentially over different types of electrodes inside capillary tubes is also evaluated. Finally, the crystal quality and the three-dimensional structure of these proteins grown with and without the electric field influence are analyzed by means of X-ray diffraction methods.

Combination of oils and gels for enhancing the growth of protein crystals

This note focuses on two different ways of enhancing the use of gels in protein crystallization by applying oils to the trials. Using a five-channel motorized syringe setup, crystals were grown in gelled microbatch drops under oil and compared with those grown under similar conditions in standard microbatch drops. The advantage of this technique over existing gel techniques is that numerous trials can be dispensed automatically, while consuming very small quantities of protein. The application of oil to improve the gel acupuncture technique was also investigated; crystal growth in the presence of an oil barrier was slower than in its absence, giving rise in each case to a single large crystal with no precipitation nor smaller crystals in the capillary.

Crystallochemical characterization of calcium oxalate crystals isolated from seed coats ofPhaseolus vulgaris and leaves ofVitis vinifera

Calcium oxalate crystals are a major biomineralization product in higher plants. Their biological function and use are not well understood. In this work, we focus on the isolation and crystallochemical characterization of calcium oxalate crystals from seed coats of Phaseolus vulgaris (prisms) and leaves of Vitis vinifera (raphides and druses) using ultrastructural methods. A proposal based on crystal growth theory was used for explaining the existence of different morphologies shown by these crystals grown inside specialized cells in plants.

Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M(r) 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of beta-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Separating nucleation and growth in protein crystallization using dynamic light scattering

A means of controlling crystallization is to separate the phases of nucleation and growth. Methods to achieve this, other than seeding, involve lowering the supersaturation by changing the temperature or diluting drops after incubating them for a given time at nucleation conditions. However, by the time nuclei or crystals are visible under the microscope too many nuclei will have formed. Dynamic Light Scattering was applied practically, to determine the most likely time for nucleation-growth decoupling to be performed successfully. The time at which DLS showed a significant change in the size-distribution of species in solution, corresponded to that optimal time.

Osteopontin Upregulation in Atherogenesis Is Associated with Cellular Oxidative Stress Triggered by the Activation of Scavenger Receptors

BACKGROUND Osteopontin (OPN) is a highly phosphorylated sialoprotein and a prominent component of mineralized extracellular matrices of bones and teeth. Although the structure of OPN has begun to be elucidated, the role of OPN overexpression in tissues distant from the bones and teeth remains poorly understood. In the present study, a rabbit model of hypercholesterolemia was employed to analyze the relationship between the vascular calcification process and OPN overexpression in the neointima of atherosclerotic plaques. METHODS OPN identification in the aorta of experimental animals fed with a high cholesterol diet was carried out by immunohistochemical procedures and Western blot analysis of tissue homogenates. Transmission electron microscopy was employed to localize target-like extracellular structures of atherosclerotic aortas. The human cell line T/G HA-VSMC was employed in the establishment of a ROS generation model employing the internalization of OxLDL particles. RESULTS Using immunohistochemical and Western blot analysis, OPN overexpression was detected in the aortas of rabbits fed a high-cholesterol diet. Results from the ultrastructural analysis of the rabbit neointima through transmission electron microscopy and from the detection of calcium phosphate precipitates by specific histochemical techniques, suggested that OPN may be functionally important as a regulator of vascular calcification. OPN was dramatically overexpressed by vascular smooth muscle cells in the presence of oxidized and acetylated LDL particles bound to scavenger receptors, thereby promoting cytosolic oxidative stress. CONCLUSIONS This study establishes the in vivo role of OPN in the intima of the aorta regulating calcium phosphate precipitate deposition in response to oxidative stress.

Turbidity as a useful optical parameter to predict protein crystallization by dynamic light scattering

Abstract The aggregation behavior of several proteins in solution including the human apolipoproteins A-II and C-III, as well as concanavalin A, thaumatin, lysozyme and mexicain, is discussed based on dynamic light scattering techniques. According to our results, the estimation of parameters such as the geometrical factor ( H ) and turbidity ( τ ) under different environmental conditions, is a useful approach in order to elucidate if protein aggregation is carried out by either nucleation or random mechanisms. We conclude that dynamic light scattering, an accurate and non-destructive technique, can be used to determine either protein precrystallization parameters or crystallization conditions when both H and τ are taken into account.

The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 α-zein

Zeins are the major storage proteins in corn seeds organized in protein bodies located in the endosperm. They are soluble in alcoholic solution and depict a high tendency to aggregation. The Z19 alpha-zein aggregates obtained by heating show a particular and interesting temperature-dependent behavior. This work was aimed at determining not only the effect of temperature on the aggregation behavior, but also the effect of the sulfhydryl groups and disulphide bonds on the thermal aggregation process under non-aqueous conditions. Z19 alpha-zein was chemically modified to obtain different sulfhydryl groups and disulphide-bonds content. Far-UV CD, ANS emission fluorescence, and dynamic light scattering, as well as differential scanning calorimetry, were performed to characterize this protein. Removal of these disulphide-bonds and alkylation of all the sulfhydryl groups in the protein promoted the lowest T(m) of 57.36 degrees C, eliminated aggregation, enhanced protein flexibility, and diminished thermal stability. These results suggest that the disulphide linkage could be the driving force for the Z19 alpha-zein aggregation.