Showing papers by "Jose A. Gavira published in 2010"

Modulation of Buried Ionizable Groups in Proteins with Engineered Surface Charge

Abstract: Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence that the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in proteins can be efficiently modulated through the rational design of the surface charge distribution, thus paving the way for the protein engineering exploitation of charge burial.

Biophysical and atomic force microscopy characterization of the RNA from satellite tobacco mosaic virus

Abstract: Agarose gel electrophoresis, circular dichroism and differential scanning calorimetry showed that single-stranded RNA from satellite tobacco mosaic virus transforms from a conformationally ‘closed state’ at 4 � C to a more conformationally ‘open state’ at 65 � C. The transition is reversible and shows no hysteresis. Atomic force microscopy (AFM) allowed visualization of the two states and indicated that the conformationally ‘closed state’ probably corresponds to the native encapsidated conformation, and that the ‘open state’ represents a conformation, characterized as short, thick chains of domains, as a consequence of the loss of tertiary interactions. Heating from 75 � Ct o 85 � C in the presence of EDTA was necessary to further unravel the ‘open’ conformation RNA into extended chains of lengths >280nm. Virus exposed to low concentrations of phenol at 65 � C, extruded RNA as distinctive ‘pigtails’ in a synchronous fashion, and these ‘pigtails’ then elongated, as the RNA was further discharged by the particles. Moderate concentrations of phenol at 65 � C produced complete disruption of virions and only remains of decomposed particles and disordered RNA were evident. AFM images of RNA emerging from disrupted virions appear most consistent with linear arrangements of structural domains.

Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels.

Abstract: The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were con­­structed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P212121, while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

Two-step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4-9.

Abstract: Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.

Toward the Crystallization of Photosystem II Core Complex from Pisum sativum L.

Abstract: The crystallization of the higher plant ∼1 gigadalton homodimeric lipid-pigment−protein complex of photosystem II constitutes a challenging undertaking that has not been crowned with success to date. In the continuation of our previous efforts (Kuta-Smatanova et al. Photosyn. Res. 2006, 90, 255−259) that had provided needle and plate crystals of plant photosystem II for the first time, we report on a study to crystallize three-dimensional crystals of the photosystem II from Pisum sativum L. suitable for X-ray analysis. In our screening, we optimized temperature, supersaturation rate, and concentrations of detergents, buffers, salts, and organic additives. We used a sitting and hanging drop vapor-diffusion method, microbatch under oil and counter-diffusion techniques, and direct and cross-influenced procedures. We have identified that the major limiting factors for the growth of crystals are the photosystem II stability and requirement of indispensable divalent cations. The newly proposed physicochemical c...