Martinez-Rodriguez S.,University of Almeria |
Martinez-Rodriguez S.,University of Granada |
Martin-Garcia J.M.,University of Almeria |
Martin-Garcia J.M.,Arizona State University |
And 5 more authors.
PLoS ONE | Year: 2014
The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging b-turn through a cluster of watermolecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation. © 2014 Bacarizo et al.
Neira J.L.,University Miguel Hernandez |
Neira J.L.,Biocomputation and Complex Systems Physics Institute |
Rizzuti B.,University of Calabria |
Iovanna J.L.,Aix - Marseille University
Archives of Biochemistry and Biophysics | Year: 2016
Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes; in humans, they are often associated with diseases. The protein NUPR1 is a multifunctional IDP involved in the development and progression of pancreatic cancer; therefore, it constitutes a target for drug design. In an effort to contribute to the understanding of the conformational features of NUPR1 and to provide clues on amino acid interactions in disordered states of proteins, we measured the pKa values of all its acidic groups (aspartic and glutamic residues, and backbone C terminus) by using NMR spectroscopy at low (100 mM) and high (500 mM) NaCl concentration. At low ionic strength, the pKa values were similar to those reported for random-coil models, except for Glu18 and Asp19, suggesting electrostatic interactions around these residues. Molecular modelling and simulation indicate an additional, significant role of nearby proline residues in determining the polypeptide conformational features and water accessibility in the region around Glu18, modulating the titration properties of these amino acids. In the other acidic residues of NUPR1, the small deviations of pKa values (compared to those expected for a random-coil) are likely due to electrostatic interactions with charged adjacent residues, which should be reduced at high NaCl concentrations. In fact, at high ionic strength, the pKa values of the aspartic residues were similar to those in a random coil, but there were still small differences for those of glutamic acids, probably due to hydrogen-bond formation. The overall findings suggest that local interactions and hydrophobic effects play a major role in determining the electrostatic features of NUPR1, whereas long-range charge contributions appear to be of lesser importance. © 2016 Elsevier Inc. All rights reserved.
Neira J.L.,University Miguel Hernandez |
Neira J.L.,Biocomputation and Complex Systems Physics Institute
Structure | Year: 2013
In this issue of Structure, Tsytlonok and colleagues describe the folding landscape of the giant HEAT-repeat protein PR65/A (a molecular adaptor of protein phosphatase 2A) by using experimental and theoretical methods. Both approaches agree in suggesting the presence of parallel folding pathways with several intermediates. © 2013 Elsevier Ltd.
Traverso J.A.,Biologia Celular y Molecular de Plantas |
Lopez-Jaramillo F.J.,University of Granada |
Serrato A.J.,Biologia Celular y Molecular de Plantas |
Ortega-Munoz M.,University of Granada |
And 6 more authors.
Journal of Plant Physiology | Year: 2010
The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRXh1) and Pisum sativum thioredoxin h2 (PsTRXh2) are both h-type TRXs from pea (Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRXh2 interacted with classical antioxidant proteins, whereas PsTRXh1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity. © 2009 Elsevier GmbH. All rights reserved.
Aguado-Llera D.,University Miguel Hernandez |
Bacarizo J.,University of Almeria |
Gregorio-Teruel L.,University Miguel Hernandez |
Taberner F.J.,University Miguel Hernandez |
And 3 more authors.
FEBS Letters | Year: 2012
Transient receptor potential (TRP) proteins are sensory-related cation channels. TRPV subfamily responds to vanilloids, generating a Ca 2+ current. TRPV1, a thermal-sensitive non-selective ion channel, possesses six transmembrane helices and the intracellular N- and C-terminal domains. The latter contains the PIP 2 and calmodulin binding sites, the TRP domain and a temperature-responding flexible region. Although the function of C-TRPV1 is known, there are no experimental reports on its structural features. Here, we describe the conformational features of C-TRVP1, by using spectroscopic and biophysical approaches. Our results show that C-TRVP1 is an oligomeric protein, which shows features of natively unfolded proteins. Structured summary of protein interactions: C-TRPV1 and C-TRPV1 bind by dynamic light scattering (View interaction) C-TRPV1 and C-TRPV1 bind by static light scattering (View interaction) C-TRPV1 and C-TRPV1 bind by cross-linking study (View interaction) C-TRPV1 and C-TRPV1 bind by comigration in non-denaturing gel electrophoresis (View interaction). © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.