Biomedical subjects

V Muñoz

Publications and source records attributed to V Muñoz.

At least 55 records · Page 3Linked to original sources

PubMed1997

Development of the multiple sequence approximation within the AGADIR model of alpha-helix formation: comparison with Zimm-Bragg and Lifson-Roig formalisms.

In this work we present the development of the multiple sequence approximation (AGADIRms) and the standard one-sequence approximation (AGADIRls) within the framework of AGADIR's alpha-helix formation model. The extensive comparison between these new formulations and the original one [AGADIR; V. Muñoz and L. Serrano (1994). Nat. Struct. Biol., Vol. 1, pp. 399-409] indicates that the standard one-sequence approximation is virtually identical to the multiple sequence approximation, while the previously used residue partition function approximation [Muñoz and Serrano (1994); (1995), J. Mol. Biol., Vol. 245, pp. 275-296] is less precise. The calculations of the average helical content performed with AGADIR are precise for peptides of less than 30 residues and progressively diverge from the multiple sequence formulation for longer peptides. The helicity distribution of heteropolypeptides with less than 50% average helical content is also well described, while those of quasi-homopolymers with high helical content tend to be-flattened. These inaccuracies lead to an underestimation of 0.017 kcal/mol for the mean-residue enthalpic contribution in AGADIR, as compared to AGADIRms and AGADIRls. The other energy contributions to alpha-helix stability are not affected by the original statistical approximation. We also discuss the particularities of the model for alpha-helix formation utilized in AGADIR and compare it with the classical Zimm-Bragg and Lifson-Roig theories. Moreover, we develop the mathematical relationships between the basic AGADIR energy contributions and helix nucleation and elongation, which permit the quantitative comparison between formalisms. Remarkably, the comparison between AGADIRms and the Lifson-Roig formalism shows that, despite the differences on treating helix/coil cooperativity, both theories give virtually identical results when an equivalent set of parameters is used. This indicates that the helix/coil transition is a solid theory independent of the particularities of the model for alpha-helix formation.

Mathematics↗

PubMed1997

Folding kinetics of Che Y mutants with enhanced native alpha-helix propensities.

In this work we study the folding kinetics of Che Y mutants in which the helical propensity of each of its five alpha-helices has been greatly enhanced by local interactions (between residues close in sequence). This constitutes an experimental test on the role of local interactions in protein folding, as well as providing new information on the details of the folding pathway of the protein Che Y. With respect to the first issue, our results show that the enhancement of helical propensities by native-like local interactions in Che Y has the following general effects: (1) the energetics of the whole Che Y folding energy landscape (folded state, intermediate, denatured state and main transition state) are affected by the enhancement of helical propensities, thus, native-like local interactions appear to have a low specificity for the native conformation; (2) our results support the idea, proposed from thermodynamic analysis of the mutants, that the denatured state under native conditions becomes more compact upon enhancement of helical propensities; (3) the rate of folding in aqueous solution decreases in all the mutants, suggesting that the optimization of the folding rate in this protein requires low secondary structure propensities. Regarding the description of the folding pathway of Che Y, we find evidence that the folding transition state of Che Y is constituted by two sub-domains with different degree of helical structure. The first includes helices 1 and 2 which are rather structured, while the second encompasses the last three helices, which are very unstructured. On the other hand, the same analysis for the folding intermediate indicates that all the five alpha-helices are, on average, rather structured. Thus, suggesting that a large structural reorganization of the last three alpha-helices must take place before folding can be completed. This conclusion indicates that the folding intermediate of Che Y is a misfolded species.

Bacterial Proteins↗

PubMed1997

Characterisation of the isolated Che Y C-terminal fragment (79-129)--Exploring the structure/stability/folding relationship of the alpha/beta parallel protein Che Y.

To gain insight into how the three-dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C-terminal 51 residues (79-129). This fragment consists of residues in the beta-strands 4 and 5 and alpha-helices 4 and 5 of native Che Y. The study has been performed by two-dimensional NMR and far-ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/ water at 273 K and 298 K. We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N-terminus observed in the native structure by over four residues belonging to the preceding loop. In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding beta-strand 4. None of the long-range NOEs present in native Che Y are observed under the explored experimental conditions. The conformational shifts of the H(alpha) protons within the alpha-helices of fragment 79-129 are identical to those of shorter synthetic peptides corresponding to the isolated alpha-helices. Thus, the fragment 79-129 appears to behave as an open chain with low local helical populations. The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N-terminal Che Y region. This is in accordance with the contact map of Che Y, which shows that the strongest non-local contacts of C-terminal residues are with residues of the N-terminal region, while those within the C-terminal region are very weak. More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C-terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.

Amino Acid Sequence↗

PubMed1997

A study of the chemical composition of Erythroxylum coca var. coca leaves collected in two ecological regions of Bolivia.

Coca-Erythroxylum coca Lamarck var. coca-remains one of the most common plants of the folk medicine of Bolivia used as a general stimulant. Aymara and Quechua natives prefer to chew the sweeter coca leaves from the Yungas (tropical mountain forests of the eastern slopes of the Andes) rather than those from the Chapare lowlands. The contents in cocaine and minor constituents of leaf samples cultivated in these regions does not rationalize this choice.

Bolivia↗

PubMed1997

Submillisecond kinetics of protein folding.

New experimental methods permit observation of protein folding and unfolding on the previously inaccessible nanosecond-microsecond timescale. These studies are beginning to establish times for the elementary motions in protein folding - secondary structure and loop formation, local hydrophobic collapse, and global collapse to the compact denatured state. They permit an estimate of about one microsecond for the shortest time in which a protein can possibly fold.

Humans↗

PubMed1996

Analysis of the effect of local interactions on protein stability.

BACKGROUND: Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.

Amino Acid Sequence↗

PubMed1996

Local versus nonlocal interactions in protein folding and stability--an experimentalist's point of view.

One of the classic important issues in protein folding and stability is the relative roles of noncovalent short-range (local) and long-range (nonlocal) interactions. Interest in this topic has been reinforced by recent developments in the analytical theory of protein folding and in lattice-based computer simulations. During the past few years, a wealth of experimental information relevant to this issue has been accumulating. In this review, we focus specifically on experimental aspects, discussing some general ideas that arise from the results obtained by many different groups using a variety of approaches. We also discuss a new experimental strategy that allows us to engineer the contribution of local interactions, and we discuss the first results obtained.

Computer Simulation↗

PubMed1996

Transjugular intrahepatic portosystemic shunt for the management of hepatic hydrothorax in the absence of ascites.

Hepatic hydrothorax in the absence of clinical ascites (HHAA) is a somewhat rare complication in decompensated cirrhosis. Patients with HHAA have a congenital defect in the tendinous portion of the diaphragm allowing rapid leakage of ascites into pleural cavity because of the cyclical negative intrathoracic pressure. Although several therapeutic approaches, including peritoneovenous shunt and chemical or surgical pleurodesis, have been attempted in these patients the results are largely unsatisfactory. A poor-risk surgical cirrhotic woman who was also considered not suitable for liver transplantation underwent transjugular intrahepatic portosystemic shunt (TIPS) for palliation of HHAA. After TIPS procedure hydrothorax completely resolved. TIPS can offer an attractive alternative to standard therapy in cirrhotic patients with HHAA.

Fatal Outcome↗

PubMed1995

Analysis of i,i+5 and i,i+8 hydrophobic interactions in a helical model peptide bearing the hydrophobic staple motif.

In this work we have analyzed by far-UV circular dichroism the contribution to alpha-helix stability of pairwise hydrophobic interactions in the hydrophobic staple motif [Muñoz et al. (1995) Nat. Struct. Biol. 2, 380-385]. For this, we have used a new series of alanine-based model peptides having a capping-box motif (Ser-X-X-Glu) and no other charged residues to facilitate the determination of the interaction energies with a helix/coil transition algorithm. Our results show that the favorable i,i+5 interaction between a hydrophobic residue (Leu, Met, Ile, Val, Phe) at position N' (before the N-cap) and a Leu at position N+4 (inside the helix) contributes up to -1.48 +/- 0.18 kcal/mol to alpha-helix stability at 278 and pH 7. More interestingly, the same hydrophobic residues at position N' interact favorably with an Ala at position N+4, although the interaction is weaker than that with Leu (up to -0.8 +/- 0.14 kcal/mol at 278 K and pH 7). To our knowledge, this is the first example in which a strong pairwise interaction with Ala is described and suggests that Ala could be less neutral in terms of side chain-side chain interactions than normally assumed. We observe a strong stereospecificity for the position N' which could be explained based on the extreme rigidity imposed by the formation in phase of the hydrophobic staple and capping-box motifs, as is seen in the protein structure database. We have also investigated the contribution to alpha-helix stability of a geometrically feasible i,i+8 hydrophobic interaction between residues N' and N+7.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1995

Structural analysis of peptides encompassing all alpha-helices of three alpha/beta parallel proteins: Che-Y, flavodoxin and P21-ras: implications for alpha-helix stability and the folding of alpha/beta parallel proteins.

In an attempt to delineate the early folding events of structurally related proteins with no sequence homology, peptides including all five alpha-helices of three alpha/beta parallel open-sheet proteins, Che-Y, flavodoxin and P21-ras, have been analyzed by circular dichroism (far-UV CD) and nuclear magnetic resonance (NMR) in water and 30% (v/v) trifluoroethanol (TFE). Comparison between the helical content estimations from far-UV CD and the results from the NMR analysis renders a reasonably good qualitative correlation, indicating that the same phenomenon is underlined by both methods. Helix limits, as indicated by the existence of (i,i + 3) nuclear Overhauser effect (NOE) cross-correlations and significant up-field conformational shifts of the C alpha H protons, are practically coincident with those in the folded protein. On the other hand, the conformation of the side-chains differs markedly from those in the folded protein. Observation of NOE cross-correlations between pairs of residues at positions i,i + 3 has been used to statistically quantify free energies of i,i + 3 side-chain-side-chain interactions between the different pairs of residues in an alpha-helix. This analysis indicates that interactions between hydrophobic side-chains seem to be quite favorable for helix formation. The behaviour in aqueous solution of the structural equivalent peptides for the three proteins is quite unrelated except for the peptides corresponding to helices two and five. We postulate that, in the alpha/beta parallel proteins, those helices that join two beta-strands flanking another non-consecutive beta-strand should not be stable for folding reasons.

Amino Acid Sequence↗

PubMed1995

Elucidating the folding problem of helical peptides using empirical parameters. II. Helix macrodipole effects and rational modification of the helical content of natural peptides.

Explaining the helical behaviour of amino acid sequences in solution could be one of the first steps in solving the protein folding problem in a rational way. The information about the conformational behaviour of helical peptides in solution, as well as the alpha-helix stability in proteins, has been utilised to derive a database with the energy contributions for various interaction taking place in an alpha-helix: intrinsic helical propensities, side-chain to side-chain interactions, main-chain to main-chain hydrogen bonds, and capping effects. This database was implemented in a algorithm based on the helix-coil transition theory (AGADIR). Here, the effects on helix stability due to interactions between charged groups and the helix macrodipole are described, quantified and implemented in AGADIR. The algorithm correctly calculates the average helical behaviour in solution of 423 peptides analysed by circular dichroism and it describes the helicity at a residue level, as found when comparing the prediction for each amino acid residue with the data derived from nuclear magnetic resonance studies. Using AGADIR we have done a rational modification of peptides corresponding to protein secondary structure elements in order to increase their helical content. The circular dichroism analysis of the mutant peptides showed a very good agreement between the experimental and calculated helical content. Moreover, in certain specific cases in which strong tertiary contacts in folded proteins do not exist, the algorithm successfully predicts the length of mutagenised alpha-helices. It is interesting to note that the final values of the parameters used do not significantly differ in absolute terms from those extracted from mutagenesis studies in proteins. This indicates that the same physico-chemical principles stand for both systems.

Algorithms↗

PubMed1995

Elucidating the folding problem of helical peptides using empirical parameters. III. Temperature and pH dependence.

Explaining the helical behaviour of amino acid sequences without tertiary interactions, in aqueous solution, could be considered one of the first steps to solve the protein folding problem in a rational way. In the accompanying paper the information about the conformational behaviour of helical peptides in solution, as well as the studies on alpha-helix stability in proteins has been utilised to derive a database of energy interactions. This database, when implemented in an algorithm based on the helix-coil transition theory (AGADIR), correctly calculates the average helical behaviour in solution of 423 peptides analysed by circular dichroism. The majority of these peptides have been studied at low temperatures (0 to 10 degrees C), and neutral pH. However, in vivo, proteins fold at higher temperatures and in some cases low or high pH values. To understand protein folding it is necessary to calculate the helical behaviour of linear peptides under very different temperature and pH experimental conditions. We have included the temperature and pH effects on the helical behaviour of peptides by means of generally accepted assumptions and simplifications. The inclusion of these terms allow us to calculate the helical behaviour of polyalanine-based peptides, as well as of complex natural sequences, under different experimental conditions.

Algorithms↗

PubMed1995

The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family.

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices. We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara). The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296). Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.

Amino Acid Sequence↗

PubMed1995

Helix design, prediction and stability.

Recent work revealing that our knowledge is now sufficient to build a reasonable quantitative model for the helix/coil transition in heteropolypeptides represents a watershed in research into alpha-helix stability, prediction and design. The opportunity is presented to design specific alpha-helix propensity patterns that may be used both to modify thermodynamic properties of target proteins and peptides, and for de novo protein design. Despite these advances, the picture is not yet complete and further studies of still poorly characterized factors are required to obtain a more precise understanding of alpha-helix stability.

Amino Acid Sequence↗

PubMed1995

The hydrophobic-staple motif and a role for loop-residues in alpha-helix stability and protein folding.

A recurrent local structural motif is described at the amino terminus of alpha-helices, that consists of a specific hydrophobic interaction between a residue located before the N-cap, with a residue within the helix (i,i+5 interaction). NMR and CD analysis of designed peptides demonstrate its presence in aqueous solution, its contribution to alpha-helix stability and its role in defining the alpha-helix N terminus limit. Comparison between the N-terminal structures of the peptide and those in proteins with the same fingerprint sequence, shows striking similarities. The change in the polypeptide chain direction produced by the motif suggests an important role in protein folding for residues located in polypeptide segments between secondary structure elements.

Amino Acid Sequence↗

PubMed1995

[Non-invasive intermittent mechanical ventilation: usefulness in treatment of chronic severe heart failure].

BACKGROUND: The higher respiratory work and less inspiratory muscle strength of patients with cardiac failure may contribute to decrease their functional capacity. AIM: To assess the effects of non invasive intermittent mechanical ventilation on clinical parameters, peripheral perfusion, cardiac and inspiratory muscle function. PATIENTS AND METHODS: Patients with chronic cardiac failure, functional capacity III-IV were subjected to 6 sessions of nasal non invasive intermittent ventilation during 4 hours or to simulated ventilation (controls). RESULTS: Fifteen ventilated patients and six controls completed the protocol. Ventilated patients improved the Mahler transition score for dyspnea by 4 +/- 1.6 points. They also improved their aerobic capacity, increasing the exercise duration from 10.9 +/- 4 to 12.7 +/- 5 min and their maximal oxygen consumption from 14.6 +/- 4 to 16.4 +/- 5.7 ml/kg/min. These patients also decreased their O2 and CO2 ventilatory equivalents. Maximal inspiratory pressure increased from 67.9 +/- 23.6 to 80.19 +/- 21.4 cm H2O, sustained maximal inspiratory pressure increased from 101.4 +/- 48 to 133 +/- 53 cm H2O and maximal endurance increased from 132 +/- 52 to 162 +/- 58 g in ventilated patients. None of these variables was modified in control patients. No changes were observed in renal function, blood volume, arterial gases, spirometry or plasma catecholamine levels in any group. CONCLUSIONS: Intermittent nasal ventilation or other measures to improve inspiratory muscle function may be beneficial for patients with severe cardiac failure.

Adult↗

PubMed1994

Helix stop and start signals in peptides and proteins. The capping box does not necessarily prevent helix elongation.

Recently, several papers have addressed the existence of helix stop signals at the beginning of alpha-helices. It has been indicated that the existence of a reciprocal backbone-side-chain hydrogen-bond interaction, designated the capping box, could be one of these signals. The fingerprint sequence of this capping box is Ser/Thr-X-X-Glu/Gln. In the fifth alpha-helix of the chemotactic alpha/beta parallel protein CheY there is such a sequence in the middle of the helix. In a peptide corresponding to this alpha-helix the capping box is bypassed, as deduced from NMR analysis. However, making the peptide shorter so that the capping box fingerprint is closer to the beginning of the peptide results in the formation of the capping box. These results indicate that, although the capping box could play a role in stabilizing and nucleating helical peptides in solution, it is not necessarily a stop signal and can be bypassed when favourable interactions exist between the surrounding residues.

Amino Acid Sequence↗

PubMed1994

Kinetic characterization of the chemotactic protein from Escherichia coli, CheY. Kinetic analysis of the inverse hydrophobic effect.

CheY, the 129 amino acid chemotactic protein from Escherichia coli, is a good model for studying the folding process of the parallel alpha/beta family of proteins. A study of the folding kinetics of CheY using fluorescence and far-UV circular dichroism (CD) stopped-flow measurements is reported. CheY has three prolines, two of them in the trans conformation and one, Pro110, with a cis Lys-Pro peptide bond. This protein presents a unimolecular, but complex, kinetic mechanism that is dominated by a slow phase compatible with a trans-cis isomerization. Mutation of Pro110 to Gly results in the disappearance of this slow phase, indicating that this cis prolyl bond is responsible for it. The slow phase is catalyzed in a very inefficient way by prolyl isomerase, indicating that the cis bond is poorly accessible to the enzyme during refolding. In agreement with this is the fact that the isomerization of the Lys109-Pro110 bond occurs in an intermediate which contains 96% of the native far-UV CD signal and 80% of the native fluorescence signal. Analysis of the unfolded protein with all its prolines in the native conformation shows the existence of a very stable intermediate in the folding reaction. Mutation of a hyperexposed hydrophobic residue, Phe14, to Asn results in an increase in the free energy of unfolding of the protein of approximately 3 kcal mol-1. Kinetic analysis of the unfolding and refolding reactions of this mutant indicates that the major stabilization effect comes from the relative destabilization of the unfolded state and the kinetic intermediate with respect to the transition state, providing kinetic evidence for the inverse hydrophobic effect. This could also indicate the existence of nonnative interactions in folding intermediates.

Bacterial Proteins↗