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M Rico

Publications and source records attributed to M Rico.

At least 19 recordsLinked to original sources
PubMed2003

Insights into the determinants of beta-sheet stability: 1H and 13C NMR conformational investigation of three-stranded antiparallel beta-sheet-forming peptides.

In a previous study we designed a 20-residue peptide able to adopt a significant population of a three-stranded antiparallel beta-sheet in aqueous solution (de Alba et al. [1999]Protein Sci.8, 854-865). In order to better understand the factors contributing to beta-sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and 13Calpha conformational shifts. The populations of the target beta-sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic beta-sheet propensities greatly disfavor beta-sheet folding and that, as already found in other beta-sheet models, specific cross-strand side chain-side chain interactions contribute to beta-sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross-strand side chain-side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor beta-sheet-forming residue, such as Gly.

Amino Acid Sequence↗
PubMed2002

Solution structure of allergenic 2 S albumins.

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower ( Helianthus annuus ), are described. The resulting common structure consists of a bundle of five alpha-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.

2S Albumins, Plant↗
PubMed2001

Three-dimensional solution structure and stability of thioredoxin m from spinach.

Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms.

Amides↗
PubMed2001

DNA-induced alpha-helical structure in the NH2-terminal domain of histone H1.

It is important to establish the structural properties of linker histones to understand the role they play in chromatin higher order structure and gene regulation. Here, we use CD, NMR, and IR spectroscopy to study the conformation of the amino-terminal domain of histone H1 degrees, free in solution and bound to the DNA. The NH(2)-terminal domain has little structure in aqueous solution, but it acquires a substantial amount of alpha-helical structure in the presence of trifluoroethanol (TFE). As in other H1 subtypes, the basic residues of the NH(2)-terminal domain of histone H1 degrees are clustered in its COOH-terminal half. According to the NMR results, the helical region comprises the basic cluster (Lys(11)-Lys(20)) and extends until Asp(23). The fractional helicity of this region in 90% TFE is about 50%. His(24) together with Pro(25) constitute the joint between the NH(2)-terminal helix and helix I of the globular domain. Infrared spectroscopy shows that interaction with the DNA induces an amount of alpha-helical structure equivalent to that observed in TFE. As coulombic interactions are involved in complex formation, it is highly likely in the complexes with DNA that the minimal region with alpha-helical structure is that containing the basic cluster. In chromatin, the high positive charge density of the inducible NH(2)-terminal helical element may contribute to the binding stability of the globular domain.

Amino Acid Sequence↗
PubMed2001

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.

Entropy↗
PubMed2001

Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity.

Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue C-terminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factor-driven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.

Amino Acid Sequence↗
PubMed2001

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family.

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.

Amino Acid Sequence↗
PubMed2001

[Clinical results of double versus single mammary artery myocardiac revascularization: 15 years of follow-up].

INTRODUCTION: Use of the left internal mammary artery to bypass the left anterior descending coronary artery reduces cardiac events and increases survival. However, there is some controversy as to the benefits of using both mammary arteries. OBJECTIVES: To assess the long-term outcome of the use of both mammary arteries in comparison with the use of only one. PATIENTS AND METHOD: A retrospective cohort study with a mean follow-up of 9.0 +/- 4.2 years was performed including 108 patients consecutively revascularized using both mammary arteries (II) and 108 patients randomly chosen in whom one mammary artery (I) was used for this purpose. RESULTS: Both groups were similar. There were no differences between the groups in operative morbidity or mortality. The survival at 10 years was similar (II: 84.61 +/- 4%; I: 85.18 +/- 3.8%), whereas recurrence of angina (II: 29.63 +/- 5.3%; I: 47.55 +/- 5.6%) (p = 0.012), the requirement for percutaneous angioplasty (II: 3.98 +/- 2%; I: 12.99 +/- 4.1%) (p = 0.009) and cardiologic events (II: 33.48 +/- 5.5%; I: 48.48 +/- 5.5%)(p = 0.022) were all lower in the group in which both mammary arteries were used. In the multivariate analysis, the use of both mammary arteries was an independent protective factor against angina recurrence (RR = 0.55), angioplasty (RR = 0.18) and cardiologic event (RR = 0.60). CONCLUSIONS: The use of both mammary arteries for revascularization does not increase operative morbidity. Since this procedure acts as an independent factor against angina recurrence, angioplasty and cardiologic event

Female↗
PubMed2001

13C(alpha) and 13C(beta) chemical shifts as a tool to delineate beta-hairpin structures in peptides.

Unravelling the factors that contribute to the formation and the stability of beta-sheet structure in peptides is a subject of great current interest. A beta-hairpin, the smallest beta-sheet motif, consists of two antiparallel hydrogen-bonded beta-strands linked by a loop region. We have performed a statistical analysis on protein beta-hairpins showing that the most abundant types of beta-hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of 13C(alpha) and 13C(beta) conformational shifts, as expected on the basis of their phi and psi angles. This fact strongly supports the potential value of 13C(alpha) and 13C(beta) conformational shifts as a means to identify beta-hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of 13C(alpha) and 13C(beta) conformational shifts in 13 short peptides, 10-15 residues long, that adopt beta-hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify beta-hairpin populations in peptides.

Carbon Isotopes↗
PubMed2001

Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin.

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man(8) glycoform and weakly to the Man(5-6) glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man(8) and -Man(5-6) glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8 )oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble.

Carbohydrate Conformation↗
PubMed2001

NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: implications for protein folding initiation.

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that approximately 25% of the residues are involved in residual structures. Conformational shifts of the alpha-protons, heteronuclear (15)N-[(1)H] NOEs and (15)N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1-70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone (1)H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.

Bacterial Proteins↗
PubMed2000

Folding kinetics of phage 434 Cro protein.

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.

Amino Acid Sequence↗
PubMed2000

Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein.

Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5-9 N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study. The conformational behavior of the protein-linked oligosaccharide Man8 is investigated on the native and on the reduced and denatured RNaseB, using a combination of NMR and theoretical calculations. Quantitative data on the NOESY crosspeaks have been obtained, thereby allowing the comparison of mobilities of homologous linkages within the glycan chain. Oligosaccharide conformations compatible with the NMR data have been explored by molecular modeling of the free oligosaccharide, using two different force fields (AMBER and SYBYL). There are some differences between the results produced by the two force fields, the AMBER simulations providing a better agreement with the experimental data. The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNase Man8 oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognition proteins and enzymes.

Magnetic Resonance Spectroscopy↗
PubMed2000

Human p8 is a HMG-I/Y-like protein with DNA binding activity enhanced by phosphorylation.

We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Amino Acid Sequence↗
PubMed2000

Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin.

The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action.

Bacterial Proteins↗
PubMed2000

Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity.

The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.

Amino Acid Sequence↗
PubMed2000

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

Allergens↗