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L Serrano

Publications and source records attributed to L Serrano.

At least 19 recordsLinked to original sources
PubMed2000

Similarities between the spectrin SH3 domain denatured state and its folding transition state.

We have expanded our description of the energy landscape for folding of the SH3 domain of chicken alpha-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on (2)H, (15)N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native alpha-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.

Acids↗
PubMed2000

Model for stathmin/OP18 binding to tubulin.

Stathmin/OP18 is a regulatory phosphoprotein that controls microtubule (MT) dynamics. The protein does not have a defined three-dimensional structure, although it contains three distinct regions (an unstructured N-terminus, N: 1-44; a region with high helix propensity, H 1: 44-89; and a region with low helix propensity, H 2: 90-142). The full protein and a combination of H 1 and H 2 inhibits tubulin polymerization, while the combination of H 1 and the N-terminus is less efficient. None of the individual three regions alone are functional in this respect. However, all of them cross-link to alpha-tubulin, but only full-length stathmin produces high-molecular-weight products. Mass spectrometry analysis of alpha-tubulin-stathmin/OP18 and its truncation products shows that full-length stathmin/OP18 binds to the region around helix 10 of alpha-tubulin, a region that is involved in longitudinal interactions in the MT, sequestering the dimer and possibly linking two tubulin heterodimers. In the absence of the N-terminus, stathmin/OP18 binds to only one molecule of alpha-tubulin, at the top of the free tubulin heterodimer, preventing polymerization.

Cloning, Molecular↗
PubMed1999

A tale of two secondary structure elements: when a beta-hairpin becomes an alpha-helix.

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the GB1 domain by a sequence with strong beta-hairpin propensity in isolation. In particular, we have chosen the sequence of the second beta-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated beta-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.

Amino Acid Sequence↗
PubMed1999

A thermodynamic analysis of a family of small globular proteins: SH3 domains.

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

Amino Acid Sequence↗
PubMed1999

Three-dimensional crystal structure of the transcription factor PhoB receiver domain.

PhoB is the response regulator of the two-component signal transduction system activated under phosphate starvation conditions. This protein is a transcription factor that activates more than 30 genes of the pho regulon and consists of two domains: a DNA binding domain and a dimerization domain, the latter being homologous to the receiver domain described for two-component response regulators. Activation by phosphorylation induces dimerization of the protein and the consequent binding to the DNA direct repeat pho box, where it promotes the binding of RNA polymerase. In the absence of phosphorylation, the activating dimerization process can be mimicked by deletion of the DNA binding domain. The three-dimensional crystal structure of the receiver domain of PhoB from Escherichia coli has been solved by multiple anomalous diffraction using a gold derivative obtained by co-crystallization, and refined using data to 1.9 A resolution. The crystal structure reveals an alpha/beta doubly wound fold, similar to other known receivers, the most conspicuous difference being the displacement of helix alpha4 towards its N terminus. The active site includes the acidic triad Asp53 (the site of phosphorylation), Asp10 and Glu9. Lys105, from loop beta5alpha5, and Glu88, from helix alpha4, interact with Asp53 via an H-bond and a water bridge, respectively. In the asymmetric unit of the crystal there are two molecules linked by a complementary hydrophobic surface, which involves helix alpha1, loop beta5alpha5 and the N terminus of helix alpha5, and is connected to the active site through the fully conserved residue Lys105 from loop beta5alpha5. The possibility that this surface is the functional surface used for the activating dimerization is discussed.

Amino Acid Sequence↗
PubMed1999

Exploring the conformational properties of the sequence space between two proteins with different folds: an experimental study.

We have examined the conformational properties of 27 polypeptides whose sequences are hybrids of two natural protein domains with 8 % sequence identity and different structures. One of the natural sequences (spectrin SH3 domain) was progressively mutated to get closer to the other sequence (protein G B1 domain), with the only constraint of maintaining the residues at the hydrophobic core. Only two of the mutants are folded, each of them having a large sequence identity with one of the two natural proteins. The rest of the mutants display a wide range of structural properties, but they lack a well-defined three-dimensional structure, a result that is not recognized by computational tools commonly used to evaluate the reliability of structural models. Interestingly, some of the mutants exhibit cooperative thermal denaturation curves and a signal in the near-ultraviolet circular dichroism spectra, both typical features of folded proteins. However, they do not have a well-dispersed nuclear magnetic resonance spectrum indicative of a defined tertiary structure. The results obtained here show that both the hydrophobic core residues and the surface residues are important in determining the structure of the proteins, and suggest that the appearance of a completely new fold from an existing one is unlikely to occur by evolution through a route of folded intermediate sequences.

Amino Acid Sequence↗
PubMed1999

Thermodynamic analysis of alpha-spectrin SH3 and two of its circular permutants with different loop lengths: discerning the reasons for rapid folding in proteins.

The temperature dependences of the unfolding-refolding reaction of a shorter version of the alpha-spectrin SH3 domain (PWT) used as a reference and of two circular permutants (with different poly-Gly loop lengths at the newly created fused loop) have been measured by differential scanning microcalorimetry and stopped-flow kinetics, to characterize the thermodynamic nature of the transition and native states. Differential scanning calorimetry results show that all these species do not belong to the same temperature dependency of heat effect. The family of the N47-D48s circular permutant (with 0-6 Gly inserted at the fused-loop) shows a higher enthalpy as happens with the PWT domain. The wild type (WT) and the S19-P20s permutant family have a more similar behavior although the second is far less stable. The crystallographic structure of the PWT shows a hairpin formation in the region corresponding to the unstructured N-terminus tail of the WT, explaining the enthalpic difference. There is a very good correlation between the calorimetric changes and the structural differences between the WT, PWT, and two circular permutants that suggests that their unfolded state cannot be too different. Elongation of the fused loop in the two permutants, taking as a reference the protein with one inserted Gly, results in a small Gibbs energy change of entropic origin as theoretically expected. Eyring plots of the unfolding and refolding semireactions show different behaviors for PWT, S19-P20s, and N47-D48s in agreement with previous studies indicating that they have different transition states. The SH3 transition state is relatively close to the native state with regard to changes in heat capacity and entropy, indicating a high degree of compactness and order. Regarding the differences in thermodynamic parameters, it seems that rapid folding could be achieved in proteins by decreasing the entropic barrier.

Amino Acid Sequence↗
PubMed1999

Association of rheumatoid arthritis with a functional chemokine receptor, CCR5.

OBJECTIVE: To investigate whether the pathogenesis of rheumatoid arthritis (RA) is associated with the functional chemokine receptor CCR5, which is the primary CC chemokine receptor expressed by T cells in rheumatoid synovium, and its nonfunctional receptor, delta32CCR5, which is generated by the homozygous 32-basepair deletion (delta32) in the CCR5 gene. METHODS: The frequency of the CCR5 genotype was compared among 673 patients with RA, 113 patients with systemic lupus erythematosus (SLE), and 815 control subjects. The CCR5 genotype was studied by polymerase chain reaction amplification of the region flanking the delta32 deletion (delta32CCR5). RESULTS: Frequencies of the wild-type CCR5 alleles (0.929, 0.907, and 0.942, respectively) and delta32CCR5 alleles (0.071, 0.093, and 0.058, respectively) in controls, SLE patients, and RA patients did not differ significantly. However, none of the RA patients had the homozygous delta32CCR5 genotype, compared with a frequency of 0.009 in controls (P = 0.014 by Fisher's exact test; chi2 = 4.12 with Yates' correction, P = 0.042) and 0.027 in SLE patients (P = 0.003 by Fisher's exact test; chi2 = 11.63 with Yates' correction, P = 0.0006). CONCLUSION: The results suggest that the CCR5 receptor plays an important role in RA and may be a suitable target for therapy.

Alleles↗
PubMed1999

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.

Crystallography, X-Ray↗
PubMed1999

Beta-hairpin and beta-sheet formation in designed linear peptides.

Recent knowledge about the determinants of beta-sheet formation and stability has notably been improved by the structural analysis of model peptides with beta-hairpin structure in aqueous solution. Several experimental studies have shown that the turn region residues can not only determine the stability, but also the conformation of the beta-hairpin. Specific interstrand side-chain interactions, hydrophobic and polar, have been found to be important stabilizing interactions. The knowledge acquired in the recent years from peptide systems, together with the information gathered from mutants in proteins, and the analysis of known protein structures, has led to successful design of a folded three-stranded monomeric beta-sheet structure.

Hydrogen Bonding↗
PubMed1999

Rational design of a GCN4-derived mimetic of interleukin-4.

In this work we describe the rational design of two helix coiled coil peptide mimetics of interleukin-4 (IL-4) which are able to recognize and bind its high affinity receptor (IL-4R alpha). We have used the leucine-zipper domain of the yeast transcription factor GCN4 as a scaffold into which the putative binding epitope of IL-4 for IL-4R alpha was transferred in a stepwise manner, using computer-aided molecular modeling. The resulting molecules bind IL-4R alpha with affinities ranging from 2 mM to 5 microM, depending on the fraction of the IL-4 binding site incorporated and on their stability. To our knowledge this is the first time a molecule capable of binding a cytokine receptor has been successfully designed in a rational manner.

Amino Acid Sequence↗
PubMed1999

The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

The protein engineering analysis of the alpha-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop beta-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Brønsted plot analysis confirms the straight phi(doubledagger-U) results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the alpha-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.

Amino Acid Sequence↗
PubMed1999

Local water bridges and protein conformational stability.

Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif--the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).

Hydrogen Bonding↗
PubMed1999

Position dependence of amino acid intrinsic helical propensities II: non-charged polar residues: Ser, Thr, Asn, and Gln.

The assumption that the intrinsic alpha-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by approximately 0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (+/-0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (+/-0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.

Amino Acid Sequence↗
PubMed1999

Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities.

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.

Amino Acids↗
PubMed1999

Stable proline box motif at the N-terminal end of alpha-helices.

We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.

Amino Acid Motifs↗
PubMed1999

Risk of thrombosis in patients with antiphospholipid antibodies and factor V Leiden mutation.

OBJECTIVE: Antiphospholipid antibodies (aPL) are thrombophilic risk markers in patients with systemic lupus erythematosus (SLE) or primary antiphospholipid syndrome (APS). The risk factors for recurrent venous or arterial thrombosis and indications for longterm anticoagulation therapy are debated. We hypothesized that carrying a second thrombophilic defect, factor V Leiden mutation, would increase the risk for thrombosis in patients with aPL. METHODS: Seventy-five patients with primary APS and 83 with SLE and aPL with or without thrombosis followed at 2 university hospitals were studied. Factor V mutation rate was analyzed in patients and in 200 healthy blood donors by polymerase chain reaction analysis. RESULTS: The prevalence of factor V Leiden mutation in patients with SLE and aPL or primary APS was similar to controls. Patients with deep vein thrombosis or arterial thrombosis did not have a significantly increased rate of factor V mutation compared to controls or to patients with aPL without thrombosis. CONCLUSION: Factor V Leiden mutation is not significantly associated with vein thrombosis in patients with aPL. However, due to the sample size we cannot rule out synergy between both factor V Leiden and aPL. A trend toward increased risk for thrombosis was detected in patients with the mutation and this should be analyzed in a larger study.

Adult↗