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L Serrano

Publications and source records attributed to L Serrano.

At least 19 recordsLinked to original sources
PubMed1995

Comparison between the phi distribution of the amino acids in the protein database and NMR data indicates that amino acids have various phi propensities in the random coil conformation.

It has been indicated that amino acids have various intrinsic phi and psi propensities, as demonstrated from the comparison between experimental secondary structure propensities and their relative statistical distribution in the protein database for the appropriate region of the Ramachandran plot. However, this does not eliminate the possibility that these experimental propensities are the result of context effects due to the secondary structure environment of the mutated position. To demonstrate that there are at least real intrinsic phi propensities, independent of context effects, we have used two different nuclear magnetic resonance parameters related to the phi dihedral angle (J3 alpha HN coupling constants and the chemical shift of the C alpha H proton), determined in random-coil tetra- and pentapeptides, and/or in proteins. Comparison of the experimentally determined values for these parameters with the theoretical ones determined from the analysis by different empirical and theoretical equations of the phi dihedral angle statistical distribution of the amino acids in the protein database, supports the idea that each amino acid has, at least, different phi intrinsic propensities. Consideration of all conformations, or only coil conformations, in the protein database produces similar results. The reasonable correlation between these experimental and theoretical data and the hydrogen-exchange data in random-coil peptides suggests that maximisation of hydrophobic surface-buried and hydrogen-bond formation with the solvent could be responsible for these different random-coil conformational preferences. Analysis of the intrinsic propensities for beta-strand, alpha-helix and polyproline II dihedral angles of the 20 amino acids in coil conformations, indicates that the side-chain of the amino acids is mainly determining the relative preferences for the phi angle.

Amino Acids↗
PubMed1995

Evidence for a two-state transition in the folding process of the activation domain of human procarboxypeptidase A2.

The activation domain of human procarboxypeptidase A2 (ADA2h), a globular open-sandwich alpha + beta domain with 80 residues and no disulfide bridges, has been studied by thermodynamic and kinetic analysis. Equilibrium denaturation by urea or temperature is fully reversible at pH 7.0 and fits to a two-state transition. The Gibbs energy of unfolding extrapolated to null concentration of chemical denaturant, delta GH2O, at pH 7.0 and 298 K, is calculated to be 17.0 +/- 1 kJ mol-1, which is within experimental error of the value determined by differential scanning calorimetry, 15.1 +/- 2 kJ mol-1. Kinetics of unfolding and refolding followed by fluorescence do not show the presence of any kinetic intermediate accumulating in the folding reaction. A value for delta GH2O of 17.9 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data. All these data indicate that the folding pathway of this domain is consistent with a two-state model (with the exception of the cis-Pro intermediates). More importantly, the analysis of this and several other small domains or proteins supports the hypothesis that stable kinetic folding intermediates are not necessary for a protein to fold. There seems to be a relationship between the size of a protein and the presence of stable kinetic intermediates. Globular proteins with less than 80 residues and no disulfide bonds follow a two-state transition, while proteins larger than 100 residues present stable kinetic folding intermediates.

Binding Sites↗
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

Experimental analysis of the Schellman motif.

Statistical analysis of the protein database indicates that the presence of a particular sequence fingerprint, involving a Gly residue at position i, two hydrophobic residues at positions i + 1 and i-4, and a polar or Ala residue at position i-2, is found at the C-terminal end of alpha-helices 5.9 times more frequently than expected from a random distribution. This particular sequence fingerprint is frequently associated (approximately 50% of the cases) with a local motif known as the Schellman motif. Formation of this motif with the above fingerprint is accompanied by an interaction between the side-chains of the two hydrophobic residues (97% of the cases). To assess the role of this hydrophobic interaction in helix stability and in the formation of the Schellman motif, we have analysed by nuclear magnetic resonance (NMR) and far-UV circular dichroism (CD) a set of polyalanine-based peptides containing the sequence fingerprint described above. Our results show that this motif is not populated to a large extent in aqueous solution and contributes little to alpha-helix stability, the opposite to what has previously been found in two local motifs at the N termini of helices (hydrophobic staple and capping-box). Addition of 30% (v/v) trifluoroethanol results in the formation of the hydrophobic interaction between residues i-4 and i + 1 of the fingerprint, thus showing that there are no sequence or sterical reasons that prevent its formation in aqueous solution. This motif could be an example of a local interaction selected both on a stability basis and because of three-dimensional packing reasons.

Amino Acid Sequence↗
PubMed1995

Side-chain interactions between sulfur-containing amino acids and phenylalanine in alpha-helices.

The side-chain-side-chain interaction between Phe residues and sulfur-containing residues (Cis and Met) in the two possible orientations at positions i, i + 4 of alpha-helices is described. We have analyzed the contribution to helical stability of the above interactions by studying eight polyalanine-based peptides differing at the residues at positions 9 and 13. These two positions were independently mutated from Ala (AA), to Cys (AC and CA), Met (AM and MA), and Phe (AF and FA) and to the pairs Phe-Met (FM), Met-Phe (MF), Phe-Cys (FC), and Cys-Phe (CF). The intrinsic helical propensities of Cys, Met, and Phe were found to be those previously described in the algorithm AGADIR. NMR analysis of the FM, MF, FC, and CF peptides showed the formation in aqueous solution of contacts between the aromatic ring and the side chains of Cys or Met, at the two i, i + 4 orientations. CD studies demonstrated the important contribution of two of these interactions (FM and FC) to alpha-helix stability (up to 2 kcal mol-1 in the Phe-Cys pair). Statistical analysis of the protein database provides a rationale for the stereospecificity and free energies of the interactions. The very favorable interaction between an aromatic ring and a sulfur-containing amino acid explains why in the protein database around 50% of the sulfur atoms are contacting aromatic rings (Reid et al., 1985).

Amino Acid Sequence↗
PubMed1995

Investigating the structural determinants of the p21-like triphosphate and Mg2+ binding site.

Amongst the superfamily of nucleotide binding proteins, the classical mononucleotide binding fold (CMBF), is the one that has been best characterized structurally. The common denominator of all the members is the triphosphate/Mg2+ binding site, whose signature has been recognized as two structurally conserved stretches of residues: the Kinase 1 and 2 motifs that participate in triphosphate and Mg2+ binding, respectively. The Kinase 1 motif is borne by a loop (the P-loop), whose structure is conserved throughout the whole CMBF family. The low sequence similarity between the different members raises questions about which interactions are responsible for the active structure of the P-loop. What are the minimal requirements for the active structure of the P-loop? Why is the P-loop structure conserved despite the diverse environments in which it is found? To address this question, we have engineered the Kinase 1 and 2 motifs into a protein that has the CMBF and no nucleotide binding activity, the chemotactic protein from Escherichia coli, CheY. The mutant does not exhibit any triphosphate/Mg2+ binding activity. The crystal structure of the mutant reveals that the engineered P-loop is in a different conformation than that found in the CMBF. This demonstrates that the native structure of the P-loop requires external interactions with the rest of the protein. On the basis of an analysis of the conserved tertiary contacts of the P-loop in the mononucleotide binding superfamily, we propose a set of residues that could play an important role in the acquisition of the active structure of the P-loop.

Amino Acid Sequence↗
PubMed1995

Folding of protein G B1 domain studied by the conformational characterization of fragments comprising its secondary structure elements.

The solution structure of the isolated fragments 1-20 (beta-hairpin), 21-40 (alpha-helix) and 41-56 (beta-hairpin), corresponding to all the secondary structure elements of the protein G B1 domain, have been studied by circular dichroism and nuclear magnetic resonance techniques. In the protein G B1-(1-20) fragment turn-like folded structures were detected in water though low populated. In the presence of 30% aqueous trifluoroethanol there is a complex conformational behaviour in which a helical structure at the N-terminal half is formed in equilibrium with random and native-like beta-hairpin structures. The peptide corresponding to the alpha-helix is predominantly unstructured in water, while in 30% trifluoroethanol it highly populates a native alpha-helical conformation, including a (i,i + 5) interaction between hydrophobic residues at its C-terminus. The third peptide was previously reported to form a monomeric native beta-hairpin structure in water [Blanco, F. J., Rivas, G. & Serrano, L. (1994a) Nature Struc. Biol. 1, 584-590]. We show in this work that the beta-hairpin structure is further stabilized in 30% trifluoroethanol and destabilised in the presence of 6 M urea, though some folded structure persists even in these highly denaturing conditions. The conformational properties of these peptides suggests that the second beta-hairpin could be an important folding initiation site on which the rest of the chain folds. Reconstitution experiments failed to show evidence of interaction between the peptides. Algorithms designed to predict the helical and extended conformations of peptides in aqueous solution successfully describe the complicated behaviour of these peptides. Comparison of the predicted and the experimental results with those for a structurally related protein, ubiquitin, shows very strong similarities, the main difference being the switch of the most stable beta-hairpin from the N-terminus in ubiquitin to the C-terminus in protein G.

Amino Acid Sequence↗
PubMed1995

The order of secondary structure elements does not determine the structure of a protein but does affect its folding kinetics.

We have analyzed the structure, stability and folding kinetics of circularly permuted forms of alpha-spectrin SH3 domain. All the possible permutations involving the disruption of the covalent linkage between two beta-strands forming a beta-hairpin have been done. The different proteins constructed here fold to a native conformation similar to that of wild-type protein, as demonstrated by nuclear magnetic resonance and circular dichroism. Although all the mutants have similar stabilities (they are 1 to 2 kcal mol-1 less stable than the wild-type) their rate constants for folding and unfolding are quite different. Protein engineering, in combination with kinetics indicates that the folding pathway has been changed in the circularly permuted proteins. We conclude that neither the order of secondary structure elements, nor the preservation of any of the beta-hairpins present in this domain, is crucial for the ability of the polypeptide to fold, but they influence the folding and unfolding kinetics and could determine its folding pathway.

Amino Acid Sequence↗
PubMed1995

Three-dimensional structure of chemotactic Che Y protein in aqueous solution by nuclear magnetic resonance methods.

The three-dimensional structure of chemotactic Che Y protein from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. A total of 20 converged structures were computed from 1545 conformationally relevant distance restraints derived from 1858 unambiguously assigned NOE cross-correlations. The resulting average pairwise root-mean-square deviation is 1.03 A for the backbone atoms and 1.69 A for all heavy atoms. If residues in the regions structurally least defined (1 to 5, 47 to 50, 76 to 79, 88 to 91 and 124 to 129) are excluded from the analysis, the root-mean-square deviations are reduced to 0.53 A and 1.23 A, respectively. The solution structure is closely similar to the refined X-ray crystal structure, except in the regions found to be less defined by NMR spectroscopy. The root-mean-square deviation between the average solution structure and the X-ray crystal structure is 0.92 A for the backbone residues (2 to 129). The highly refined solution structure determined herewith provides an essential background to delineate functionally important conformational changes brought about by different effectors.

Bacterial Proteins↗
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

Empirical correlation for the replacement of Ala by Gly: importance of amino acid secondary intrinsic propensities.

A series of Ala vs. Gly mutations at different helical and nonhelical positions of the chemotactic protein CheY, from E. coli, has been made. We have used this information to fit a general analytical equation that describes the free energy changes of an Ala to Gly mutation within +/- 0.45 kcal mol-1 with 95% confidence. The equation includes three terms: (1) the change in solvent-accessible hydrophobic surface area, corrected for the possible closure of the cavity left by deleting the C beta of the Ala; (2) the change in hydrophilic area of the nonintramolecularly hydrogen-bonded groups; and (3) the dihedral angles of the position being mutated. This last term extends the calculation to any conformation, not only alpha-helices. The general applicability of the equation for Ala vs. Gly mutations, when Ala or a small solvent-exposed polar residue is the wild-type residue, has been tested using data from other proteins: barnase, CI2 trypsin inhibitor, T4 lysozyme, and Staphylococcus nuclease. The predictive power of this simple approach offers the possibility of extending it to more complex mutations.

Alanine↗
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↗
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↗