80 research outputs found
High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution
Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H···H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico
High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution
Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H···H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico
Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation
BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays
Characterization of the thermal and photoinduced reactions of photochromic spiropyrans in aqueous solution
Six water-soluble spiropyran derivatives have been characterized with respect to the thermal and photoinduced reactions over a broad pH-interval. A comprehensive kinetic model was formulated including the spiro- and the merocyanine isomers, the respective protonated forms, and the hydrolysis products. The experimental studies on the hydrolysis reaction mechanism were supplemented by calculations using quantum mechanical (QM) models employing density functional theory. The results show that (1) the substitution pattern dramatically influences the pKa-values of the protonated forms as well as the rates of the thermal isomerization reactions, (2) water is the nucleophile in the hydrolysis reaction around neutral pH, (3) the phenolate oxygen of the merocyanine form plays a key role in the hydrolysis reaction. Hence, the nonprotonated merocyanine isomer is susceptible to hydrolysis, whereas the corresponding protonated form is stable toward hydrolytic degradation
Education and inequality in Finland, Spain and Brazil
Production of INCASI Project H2020-MSCA-RISE-2015 GA 691004Finland, Spain and Brazil are three very internally complex and heterogeneous realities, with contradictions and permanent reforms to their education systems. In a first quantitative approach each country can be placed in a continuum of the education system that goes from most successful in terms of reaching a high level of education all across the population, in conditions of equity and facilitating youths' incorporation into the labour market, to least successful, with Finland and Brazil occupying either end of the spectrum respectively and Spain occupying an intermediate situation. Although there are differences, they share certain tensions in their respective education systems. On the one hand, about the conception of education, ranging from more utilitarian, human capital theories, to the more humanist and civic-minded perspective. On the other hand, the challenge of comprehensiveness between an academic and a vocational path. In addition, there is also the challenge of improving the education level of the population while also improving equality. The tensions differ from country to country, since their education traditions and cooperation and conflict strategies between the education agents, with varying levels of resources and different alliances with political actors vary, as does the social consensus
Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library
Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue
Optimization of Enzymatic Biochemical Logic for Noise Reduction and Scalability: How Many Biocomputing Gates Can Be Interconnected in a Circuit?
We report an experimental evaluation of the "input-output surface" for a
biochemical AND gate. The obtained data are modeled within the rate-equation
approach, with the aim to map out the gate function and cast it in the language
of logic variables appropriate for analysis of Boolean logic for scalability.
In order to minimize "analog" noise, we consider a theoretical approach for
determining an optimal set for the process parameters to minimize "analog"
noise amplification for gate concatenation. We establish that under optimized
conditions, presently studied biochemical gates can be concatenated for up to
order 10 processing steps. Beyond that, new paradigms for avoiding noise
build-up will have to be developed. We offer a general discussion of the ideas
and possible future challenges for both experimental and theoretical research
for advancing scalable biochemical computing
Entre luzes e sombras: o passado imediato e o futuro possível da pesquisa em juventude no Brasil
Crystal structure of the aldehyde dehydrogenase from Thermus thermophilus at 2.2 Ǻ resolution
Crystal structure of the aldehyde dehydrogenase from Thermus thermophilus at 2.2 Ǻ resolution
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