Aguado-Llera D.,University Miguel Hernandez |
Domenech R.,University Miguel Hernandez |
Marenchino M.,Spectroscopy and NMR Unit CNIO |
Vidal M.,CSIC - Biological Research Center |
And 2 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2012
E2 ubiquitin conjugases are ∼ 20 kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-Cca was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-Cca towards non-canonical E3 ligases. Binding of E2-Cca to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ∼ 400 nM, as shown by fluorescence and ITC. Furthermore, binding of E2-C ca to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-Cca comprises the first β-strand and nearby residues. © 2012 Elsevier B.V. All rights reserved.
Predicting stabilizing mutations in proteins using Poisson-Boltzmann based models: Study of unfolded state ensemble models and development of a successful binary classifier based on residue interaction energies
Estrada J.,University of Zaragoza |
Estrada J.,Biocomputation and Complex Systems Physics Institute BIFI |
Echenique P.,Biocomputation and Complex Systems Physics Institute BIFI |
Echenique P.,CSIC - Institute of Physical Chemistry "Rocasolano" |
And 3 more authors.
Physical Chemistry Chemical Physics | Year: 2015
In many cases the stability of a protein has to be increased to permit its biotechnological use. Rational methods of protein stabilization based on optimizing electrostatic interactions have provided some fine successful predictions. However, the precise calculation of stabilization energies remains challenging, one reason being that the electrostatic effects on the unfolded state are often neglected. We have explored here the feasibility of incorporating Poisson-Boltzmann model electrostatic calculations performed on representations of the unfolded state as large ensembles of geometrically optimized conformations calculated using the ProtSA server. Using a data set of 80 electrostatic mutations experimentally tested in two-state proteins, the predictive performance of several such models has been compared to that of a simple one that considers an unfolded structure of non-interacting residues. The unfolded ensemble models, while showing correlation between the predicted stabilization values and the experimental ones, are worse than the simple model, suggesting that the ensembles do not capture well the energetics of the unfolded state. A more attainable goal is classifying potential mutations as either stabilizing or non-stabilizing, rather than accurately calculating their stabilization energies. To implement a fast classification method that can assist in selecting stabilizing mutations, we have used a much simpler electrostatic model based only on the native structure and have determined its precision using different stabilizing energy thresholds. The binary classifier developed finds 7 true stabilizing mutants out of every 10 proposed candidates and can be used as a robust tool to propose stabilizing mutations. © 2015 the Owner Societies.
Galvez J.A.,University of Zaragoza |
Diaz-De-Villegas M.D.,University of Zaragoza |
Alias M.,University of Zaragoza |
Alias M.,Biocomputation and Complex Systems Physics Institute BIFI |
Badorrey R.,University of Zaragoza
Journal of Organic Chemistry | Year: 2013
Silver-catalyzed endo-selective and copper-catalyzed exo-selective asymmetric [3 + 2] cycloadditions of acrylates to chiral iminoesters derived from d-glyceraldehyde have been investigated. The reaction diastereoselectively provides highly functionalized pyrrolidines. This approach was used to develop the first asymmetric synthesis of a key intermediate in the synthesis of pyrrolidine influenza neuramidinase inhibitors. © 2013 American Chemical Society.