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Neira J.L.,University Miguel Hernandez | Neira J.L.,Institute Biocomputacion Y Fisica Of Sistemas Complejos | Sevilla P.,Complutense University of Madrid | Sevilla P.,CSIC - Institute for the Structure of Matter | Garcia-Blanco F.,Complutense University of Madrid
Physical Chemistry Chemical Physics | Year: 2012

The α-splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is involved in protein-biomolecule interactions. The conformational stability of SAMp73 is low (∼5 kcal mol-1), although its thermal stability is high. To explain this high thermostability, we studied the dynamics of SAMp73 over a wide range of GdmCl (guanidine hydrochloride) concentrations and temperatures by NMR relaxation, NMR hydrogen-exchange (HX) and fluorescence lifetime approaches. The slowest exchanging residues of SAMp73 belong to the helical regions, and they did exchange by a global unfolding process. Moreover, SAMp73 was very flexible, with most of its amide protons affected by slow μs-ms conformational exchange. Within this time scale, the residues of SAMp73 with the largest exchange rates (Rex) were involved in binding with other molecules; therefore, the flexibility in the μs-ms range was associated with biological functions. As the [GdmCl] increased, the pico-to-nanosecond flexibility of the backbone amide protons raised, but it did so differently depending on the residue. We were able to obtain, for the first time, the linear [GdmCl]-variation of the local conformational entropies, mSi, which ranged from 5.3 to 0.3 cal mol-1 K-1 M-1, similar to those measured by using macroscopic techniques in other proteins. Conversely, the temperature dependence of the pico-to-nanosecond dynamics of the backbone amide protons of SAMp73 indicates that the flexibility of some residues decreased with the temperature; these results explain the high thermostability of the protein. © 2012 The Owner Societies.

Neira J.L.,University Miguel Hernandez | Neira J.L.,Institute Biocomputacion Y Fisica Of Sistemas Complejos
Archives of Biochemistry and Biophysics | Year: 2014

The alpha splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, is involved in lipid binding and it is thought to mediate in protein-protein interactions. SAMp73 is composed of five helices (α1-α5). In this work, we dissected SAMp73 in fragments encompassing the different helices, to study the conformational stability of the isolated elements of secondary structure. There was no evidence of stable residual helical structure in the isolated α1, α4 and α5 helices in aqueous solution, as shown by 2D-1H NMR and far-UV CD spectroscopies; those helices acquired native-like helical structure in the presence of 40% trifluoroethanol (TFE). The population of helical structure in α5 seemed to be driven by the indole moiety of Trp542, and it was enhanced by the presence of α4. On the other hand, helices α2 and 310(α3) had a tendency to self-associate even in TFE-water solutions. However, the short, aggregation-prone 310(α3) helix was key to attain the native-like fold of SAMp73, as suggested by experiments with non-covalent complexes among the peptides. © 2014 Elsevier Inc. All rights reserved.

Neira J.L.,University Miguel Hernandez | Neira J.L.,Institute Biocomputacion Y Fisica Of Sistemas Complejos
Archives of Biochemistry and Biophysics | Year: 2013

NMR spectroscopy is one of the few biophysical methods that can provide atomic-level insight into the conformation of partially folded states and/or intermediates present along the protein folding pathway. Such studies are important not only within the context of the protein folding problem, but also to push forward the technique, due to the challenging nature of the systems studied. In fact, new NMR methods have been created, and applied, in an attempt to characterize the conformational features of the states along the folding pathway. Describing the structures along the folding landscape is of key importance to comprehend the folding reaction, design new proteins and to understand how several polypeptide chains are implicated in pathogenic amyloid states. The last advances in several approaches, which use NMR: (i) to monitor the protein folding pathway and/or, (ii) to characterize the structure of the intermediate states in such reaction are reviewed in this work. © 2012 Elsevier Inc. All rights reserved.

Saelices L.,University of Seville | Galmozzi C.V.,University of Seville | Florencio F.J.,University of Seville | Muro-Pastor M.I.,University of Seville | And 2 more authors.
Biochemistry | Year: 2011

In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K D ∼1 μM) than that measured for IF7 (∼0.3 μM). The K Ds determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system. © 2011 American Chemical Society.

Benavides-Riveros C.L.,University of Zaragoza | Benavides-Riveros C.L.,Institute Biocomputacion Y Fisica Of Sistemas Complejos | Toranzo I.V.,University of Granada | Dehesa J.S.,University of Granada
Journal of Physics B: Atomic, Molecular and Optical Physics | Year: 2014

The ground-state entanglement of a single particle of the N-harmonium system (i.e., a completely integrable model of N particles where both the confinement and the two-particle interaction are harmonic) is shown to be analytically determined in terms of N and the relative interaction strength. For bosons, we compute the von Neumann entropy of the one-body reduced density matrix by using the corresponding natural occupation numbers. A critical number, Nc, of particles exists, and below it, for positive values of the coupling constant, the entanglement grows when the number of particles increases; the opposite occurs for . For fermions, we compute the one-body reduced density matrix for the closed-shell spinned case. In the strong coupling regime, the linear entropy of the system decreases when N grows. For fixed N, the entanglement is found (a) to decrease (increase) for negatively (positively) increases values of the coupling constant, and (b) to grow when the energy increases. Moreover, the spatial and spin contributions to the total entanglement are found to be of comparable size. © 2014 IOP Publishing Ltd.

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