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Miquel Pons

Publications and source records attributed to Miquel Pons.

At least 19 recordsLinked to original sources
PubMed2007

Influence of the Hofmeister anions on protein stability as studied by thermal denaturation and chemical shift perturbation.

The influence of external cosolutes on the thermal stability of the B1 domain of protein L (ProtL) has been studied by circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The thermal denaturation midpoint is effectively modulated by the addition of a suite of anions and follows the Hofmeister series. The maximum increase in thermostability (corresponding to 14 degrees C) was observed in the presence of 1 M sodium sulfate. After conversion of the experimental data into the change in the virial coefficient, a mechanistic model was used to estimate the relative contributions from excluded volume and preferential anion solvation for each anion. As expected, the excluded volume term stabilizes the native conformation of ProtL for all the cosolutes, but opposite effects on protein stability arise from the anion's solvation depending on their tendency to interact with or to become excluded from the protein surface. This behavior is in agreement with the results of independent NMR experiments: the anions that strongly interact with the protein surface produce significant perturbations in the amide protein chemical shift (delta d23(HN)). A correlation obtained between delta d23(HN) and the temperature coefficients for the different amide protons provides qualitative information about the structural determinants for the interaction between the protein surface and the cosolute.

Anions↗
PubMed2006

Fast 2D NMR ligand screening using Hadamard spectroscopy.

Fast 2D NMR-based screening can be achieved using Hadamard encoded spectroscopy to focus on the signals of interest (e.g., enzyme active or ligand recognition sites). By recording a set of Hadamard spectra (a "Hadamard constellation") with relative offsets comparable to the excitation bandwidth, quantitative ligand-induced shifts can be obtained from peak intensities.

Carrier Proteins↗
PubMed2006

New roles for key residues in helices H1 and H2 of the Escherichia coli H-NS N-terminal domain: H-NS dimer stabilization and Hha binding.

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.

Amino Acid Sequence↗
PubMed2005

On the origin of the thermostabilization of proteins induced by sodium phosphate.

The B1 domain of protein L shows a linear rise in thermostability with increasing concentrations of sodium phosphate. Equal behavior is observed for a set of mutant proteins where surface lysines are mutated to noncharged residues, but the mutant's thermostabilities show different sensitivities to phosphate, encoded in the varying slopes observed (mi). The melting temperature in the absence of the cosolute also correlates linearly with mi. The stabilizing effect of the phosphate ion reaches a saturation point, which has been experimentally determined for protein L (1610 mM phosphate). These results indicate that the phosphate-induced stabilization is an inherent property of the protein, encoded in the amino acid sequence. Changes in stability upon mutation are attributed to a redistribution of the overall network of solvated surface charges. Stabilization by phosphate is understood in terms of interactions with the protein surface, reducing the unfavorable contacts between like charges, maximizing the number of accessible conformations of the surface-charged side chains, and optimizing solvation.

Bacterial Proteins↗
PubMed2005

Interaction between the bacterial nucleoid associated proteins Hha and H-NS involves a conformational change of Hha.

The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 degrees C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS.

Amino Acid Sequence↗
PubMed2004

Evaluation of chiral recognition ability of a novel uranyl-salophen-based receptor: an easy and rapid testing protocol.

A novel member of a new class of chiral uranyl-salophen complexes has been synthesised. The chiral recognition ability of this receptor toward the enantiomers of two primary amines, a sulfoxide, and a quaternary ammonium chloride has been evaluated for the first time. The enantioselectivities obtained are encouraging. The NMR method developed for this purpose allows a fast, quantitative determination of the enantioselectivity of the host directly from its racemic mixture and could find application as a preliminary screening tool in the search for new receptors using combinatorial methods. The experiments carried out in this context demonstrated also that the activation barrier for the racemisation of such chiral uranyl-salophen receptors is much higher than the lower limit of 21 kcal mol(-1) previously reported.

Benzophenones↗
PubMed2004

A new class of foldamers based on cis-gamma-amino-L-proline.

A synthetic method for the preparation of conformationally constrained gamma-peptides derived from gamma-amino-L-proline is described. The methodology allows the independent buildup of the peptide backbone and the introduction of sequential variations by reactions with the alpha-amino group of gamma-aminoproline. Both alkyl- and acyl-substituted gamma-peptides have been prepared and studied by CD and NMR. Conformational restrictions due to the cyclic structure of the monomer give rise to long-range interactions that are indicative of secondary structures even in aqueous solution. Interresidue NOEs suggest a concatenation of turns that, in a permissive solvent, could give rise to an isolated hydrogen bond ribbon, flanked and protected by proline rings.

Aminobutyrates↗
PubMed2004

Interpretation of NMR relaxation properties of Pin1, a two-domain protein, based on Brownian dynamic simulations.

Many important proteins contain multiple domains connected by flexible linkers. Inter-domain motion is suggested to play a key role in many processes involving molecular recognition. Heteronuclear NMR relaxation is sensitive to motions in the relevant time scales and could provide valuable information on the dynamics of multi-domain proteins. However, the standard analysis based on the separation of global tumbling and fast local motions is no longer valid for multi-domain proteins undergoing internal motions involving complete domains and that take place on the same time scale than the overall motion. The complexity of the motions experienced even for the simplest two-domain proteins are difficult to capture with simple extensions of the classical Lipari-Szabo approach. Hydrodynamic effects are expected to dominate the motion of the individual globular domains, as well as that of the complete protein. Using Pin1 as a test case, we have simulated its motion at the microsecond time scale, at a reasonable computational expense, using Brownian Dynamic simulations on simplified models. The resulting trajectories provide insight on the interplay between global and inter-domain motion and can be analyzed using the recently published method of isotropic Reorientational Mode Dynamics which offer a way of calculating their contribution to heteronuclear relaxation rates. The analysis of trajectories computed with Pin1 models of different flexibility provides a general framework to understand the dynamics of multi-domain proteins and explains some of the observed features in the relaxation rate profile of free Pin1.

Anisotropy↗
PubMed2003

Peptide binding induces large scale changes in inter-domain mobility in human Pin1.

Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.

Amino Acid Motifs↗
PubMed2003

Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution.

We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse (15)N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide (15)N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.

Animals↗
PubMed2003

Sucralfate therapy in NSAID bleeding gastropathy.

BACKGROUND AND AIMS: A randomized, double-blind, placebo-controlled, multicenter study was conducted to assess the efficacy of 2 g sucralfate suspension in treating gastric mucosal lesions caused by long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: Only patients given NSAIDs continuously for at least 2 months with positive fecal occult blood (FOB) and endoscopically confirmed mild to moderate mucosal lesions (Lanza scale, grades 2-4) were included. After 1-week run-in phase, patients were stratified into 2 groups according to gastropathy-related symptoms during the preceding 7 days (symptomatic vs. asymptomatic) and randomized to 2 g (10 mL) of sucralfate suspension or placebo twice a day over a 6-week period. NSAIDs were given according to each patient's dosage schedule and always after meals. RESULTS: Twenty-five patients received sucralfate and 25 received placebo. At the end of the study, 68% (17/25) of patients given sucralfate had no lesions (Lanza grade 0) on endoscopy compared with 35% (8/23) in controls (P = 0.042). The Lanza grades in patients given sucralfate were significantly improved compared with the placebo patients (P = 0.022). CONCLUSIONS: In this target population selected according to positive FOB test and endoscopic evidence of mucosal injury, chronic administration of sucralfate significantly decreased NSAID-induced gastric erosions.

Adult↗
PubMed2003

Subtalar arthroereisis for posterior tibial tendon dysfunction: a preliminary report.

BACKGROUND: The purpose of this study was to evaluate preliminary results with Kalix subtalar arthroereisis in sinus tarsi for stage II posterior tibial tendon dysfunction. METHODS: Twenty-one patients with stage II posterior tibial dysfunction, according to Johnson and Storm, underwent surgical treatment between July 1999 and December 2000. All patients were evaluated clinically using the America Orthopaedic Foot and Ankle Society (AOFAS) hindfoot-ankle score. We performed a tendon repair depending on the type and location of the injury and implanted a Kalix endorthesis in the sinus tarsi. RESULTS: Nineteen patients attended for clinical review with an average follow-up of 27.31 months (range, 19-36). AOFAS scale improved from a preoperative average of 47.2 to an average of 81.6 at revision. The most important improvement was observed in pain (16.3 preoperative to 31.6 postoperative). Two cases required removal of the endorthesis for pain, probably because the endorthesis was too big, without any loss of correction. Patient satisfaction was "satisfied" or "very satisfied" in 17/19. All except three patients would have elected to undergo the same procedure. CONCLUSIONS: Subtalar arthroereisis by means of implantation of a Kalix endorthesis in the sinus tarsi, with prior correction of the deformity and tendon repair, offers an alternative to bone operations such as calcaneal osteotomies, lengthening the external column, or arthodesis in patients with stage II posterior tibial dysfunction.

Adult↗
PubMed2002

Peptide dendrimers based on polyproline helices.

We present a new family of peptide dendrimers based on polyproline helices and cis-4-amino-L-proline as a branching unit. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational transition between polyproline type I helix and polyproline type II helix was observed by circular dichroism in branched polyproline building blocks with more than 14 proline residues and in the resulting dendrimers. Both linear and dendritic polyprolines were found to be actively internalized by rat kidney cells. Preliminary results show that the antibiotic ciprofloxacin form complexes with branched polyproline chains in 99.5% propanol.

Animals↗
PubMed2002

NMR measurement of the off rate from the first calcium-binding site of the synaptotagmin I C2A domain.

The off rate from the first calcium-binding site of the C2A domain of synaptotagmin I, a putative calcium receptor in neurotransmitter release, has been determined by 15N-nuclear magnetic resonance relaxation dispersion measurements. The exchange rate was obtained by fitting the dependence of the transverse relaxation rates on the interval between 180 degrees pulses in relaxation-compensated CPMG experiments at 3.2 microM calcium concentration. The measured k(ex) is 2.0x10(3) x s(-1). The calcium on rate of 3.5+/-1x10(7) x s(-1), determined from the measured off rate and the dissociation constant (5.3x10(-5) M), is close to the diffusion limit. These results are consistent with the proposed role of synaptotagmin I as a calcium sensor in release, but suggest that additional factors may help to accelerate the diffusion of Ca2+ to the sensor.

Binding Sites↗
PubMed2002

Lanthanide modulation of the orientation of macromolecules induced by purple membrane.

Addition of Tb3+ to purple membrane (PM) suspensions changes the orientation of the menbrane normal from parallel to perpendicular with respect to the magnetic field. Residual dipolar couplings measured in protein L in the presence of PM are scaled by a factor of -1/2. NMR line broadening and cross-correlation effects induced by the addition of PM are partially reversed by Tb3+ but not by Tm3+ that has no effect on the orientation of PM. This is interpreted as the result of anisotropically restricted motion of protein L in the transiently PM-bound form.

Bacterial Proteins↗
PubMed2002

Structural, kinetic and cytotoxicity aspects of 12-28 beta-amyloid protein fragment: a reappraisal.

A chemical, structural and biological study on the beta-amyloid peptide beta12-28 is reported which was carried out in order to assess the feasibility using this peptide fragment as a model of the natural beta-amyloid protein. The aggregation properties of beta12-28 have been investigated by pulse field-gradient NMR spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. The results obtained suggest that beta12-28 behaviour is comparable to that of the natural beta-amyloid protein although kinetically slower. Translational diffusion coefficients obtained by NMR on an aged beta12-28 solution suggest that the soluble peptide fraction is composed of oligomeric intermediates adopting an extended ellipsoidal assembly rather than a spherical one. The beta12-28 peptide proved to be cytotoxic in PC12 cell cultures as monitored by the MTT assay, although a lack of reproducibility was observed in the dose-response experiments.

Amino Acid Sequence↗