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L Serrano

Publications and source records attributed to L Serrano.

At least 91 records · Page 5Linked to original sources
PubMed1997

Role of a nonnative interaction in the folding of the protein G B1 domain as inferred from the conformational analysis of the alpha-helix fragment.

BACKGROUND: The role of local interactions in protein folding and stability can be investigated by the conformational analysis of protein fragments. The hydrophobic staple and Schellman motifs have been described at the N and C terminus, respectively, of protein alpha-helices. These motifs are characterized by an interaction between two hydrophobic residues, one outside the helix and one within the helix, and their importance for helix stability has been analyzed in model peptides. In the alpha-helix of the protein G B1 domain, only the Schellman motif is formed--the hydrophobic staple motif is absent despite the favourable sequence pattern. We have experimentally analyzed the solution conformation of the 19-41 fragment of protein G. This peptide comprises the helical residues and contains both the hydrophobic staple and Schellman motif sequences. RESULTS: In the isolated peptide in water, the hydrophobic staple motif is formed and stabilizes the helical structure as compared with a shorter peptide lacking it, but the Schellman motif is not formed. In 30% aqueous TFE, the helix is more stable than in pure water and both motifs are formed. CONCLUSIONS: The results suggest that the importance of each motif for the folding and stability of protein G is different. The nonnative hydrophobic staple interaction can help to nucleate the helix at the beginning of folding but has later to be disrupted. The Schellman motif, while not providing enough energy for substantial helix stabilization in the unfolded state, could be important for determining the local fold of the sequence in the context of the rest of the protein.

Amino Acid Sequence↗
PubMed1997

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: comparison of unrefined and refined structure sets with the crystal structure.

The assignment of the 1H and 15N nuclear magnetic resonance spectra of the Src-homology region 3 domain of chicken brain alpha-spectrin has been obtained. A set of solution structures has been determined from distance and dihedral angle restraints, which provide a reasonable representation of the protein structure in solution, as evaluated by a principal component analysis of the global pairwise root-mean-square deviation (rmsd) in a large set of structures consisting of the refined and unrefined solution structures and the crystal structure. The solution structure is well defined, with a lower degree of convergence between the structures in the loop regions than in the secondary structure elements. The average pairwise rmsd between the 15 refined solution structures is 0.71 +/- 0.13 A for the backbone atoms and 1.43 +/- 0.14 A for all heavy atoms. The solution structure is basically the same as the crystal structure. The average rmsd between the 15 refined solution structures and the crystal structure is 0.76 A for the backbone atoms and 1.45 +/- 0.09 A for all heavy atoms. There are, however, small differences probably caused by intermolecular contacts in the crystal structure.

Amino Acid Sequence↗
PubMed1997

Loop length, intramolecular diffusion and protein folding.

Intramolecular diffusion plays a role in protein folding as shown by kinetic experiments on two alpha-spectrin SH3 domain circular permutants (S19-P20s and N47-D48s), with different poly-glycine loop lengths. Insertion of up to 10 Gly residues does not alter the structure of the folded state nor the overall characteristics of the denatured ensemble. The apparent level of the energy barrier between the denatured and folded species increases linearly with the number of inserted glycines. This suggests that the transition state itself and/or possibly previous transient unstable intermediates are accessed with more difficulty when loop length is increased. The fact that the induced impediment is directly proportional to the number of Gly residues and not to the free energy difference in the folded state indicates that diffusion of different parts of the molecule relative to each other is taking place on going from the denatured ensemble to the transition state. Our results also suggest that transition state ensembles could be more homogenous than recently postulated.

Amino Acid Sequence↗
PubMed1997

Apoptosis and proliferation of fibroblasts during postnatal skin development and scleroderma in the tight-skin mouse.

Tight-skin (Tsk) is a dominant gene mutation that causes a fibrotic skin disease in mice, similar to human scleroderma. Both conditions are characterized by increased numbers of dermal fibroblasts containing high levels of procollagen mRNA. Whether this fibroblast population arises from fibroblast growth or fibroblast transcriptional activation is debated. Proliferation and apoptosis of fibroblasts of normal and Tsk mice were studied in skin sections before, at onset, and in established fibrosis. Tissues sections were immunostained with proliferating cell nuclear antigen (PCNA) as proliferation marker. Apoptosis was investigated by in situ end-labeling of fragmented DNA and nuclear staining with propidium iodide. The expression of the apoptosis inhibitor Bcl-2 was investigated by immunohistochemistry. We demonstrate differences in fibroblast proliferation and apoptosis related to postnatal skin growth and development. Neonatal skin exhibits the highest levels of proliferation and apoptosis in fibroblasts. In contrast, low proliferation and absence of apoptosis characterizes adult fibroblasts. Skin fibroblasts express Bcl-2 only in newborns, and at other ages Bcl-2 was restricted to epithelial cells. Our results also suggest that neither increased fibroblast proliferation nor defective apoptosis accounts for the fibrotic phenotype of Tsk. Therefore, transcriptional activation of extracellular matrix genes appears more relevant in the pathogenesis of Tsk fibrosis.

Animals↗
PubMed1997

Diagnostic and therapeutic thoracoscopy in esophageal cancer.

The decision before operation whether an esophagus affected by cancer can be resected is difficult. In order to determine if the tumor can be removed or not, patients must undergo surgery. This high risk intervention is often carried out only to prescribe palliative treatment. We used a thoracoscopic technique for the diagnosis and resection of esophageal cancer with the aim of improving the high morbidity rate associated with esophageal open surgery. A right thoracoscopy was performed in seven patients and only four underwent resection. The entire esophagus was mobilized thoracoscopically and the intervention was completed through the abdomen and the neck. In three patients in whom resection was not considered possible, the thoracoscopic procedure was done with excellent tolerance, and treated by palliative methods. Thoracoscopy is a very valuable procedure for the diagnosis of resectability as well as for the mobilization of the esophagus prior to resection.

Endoscopes↗
PubMed1996

Rational design of specific high-affinity peptide ligands for the Abl-SH3 domain.

SH3 domains bind proline-rich peptides with affinities in the order of 0.2-50 microM. In general, these domains are quite promiscuous, and the same peptide can bind to several different SH3 domains with similar affinities (i.e., 3BP1 peptide to Abl- and Fyn-SH3). This poor affinity and specificity make it difficult to elucidate their role in vivo as well as the use of peptides to specifically bind to a single domain. Here, we report that by using existing biocomputing tools, as well as simple physicochemical reasoning, it is possible to design mutations in the 3BP1 peptide (Met4-Tyr, Pro5-Ser, and Leu8-Pro), so that the affinity for Abl-SH3 increases 20-fold (p40 peptide: APTYSPPPPP; Kd = 0.4 microM), while that for the closely related domain, Fyn-SH3, decreases 10-fold. Both the RT and n-Src loops are responsible for regulating the specificity for Pro-rich ligands and more specifically residues Ser15, Thr19, and Glu38 in Abl-SH3. The first six positions in the 3BP1 peptide are important for determining the specificity for SH3 domains, while the remaining four seem to be more important for the affinity. Moreover, by choosing rationally the substituents, it is possible to replace some of the Pro residues postulated to be essential for the interaction with SH3 domains and still have a significant affinity. This indicates that the sequence repertoire that could interact with a specific SH3 domain could be larger than previously thought.

Amino Acid Sequence↗
PubMed1996

Infrared evidence of a beta-hairpin peptide structure in solution.

The IR spectrum of an 16-amino acid peptide corresponding, according to NMR studies, to a beta-hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel beta-sheet: the low-frequency band that in a beta-sheet structure is located at approximately 1632 cm-1 appears here at approximately 1620 cm-1, and the high-frequency component does not undergo the isotopic shift typical of beta-sheet from 1690 to 1675 cm-1 when transferred to D2O. The infrared characteristics associated with beta-hairpins have been described so far in two proteins, in one of which, whose three-dimensional structure is known from X-ray diffraction, a beta-hairpin has actually been detected.

Bacterial Proteins↗
PubMed1996

The three-dimensional structure of two mutants of the signal transduction protein CheY suggest its molecular activation mechanism.

The three-dimensional crystal structures of the single mutant M17G and the triple mutant F14G-S15G-M17G of the response regulator protein CheY have been determined to 2.3 and 1.9 angstrom, respectively. Both mutants bind the essential Mg2+ cation as determined by the changes in stability, but binding does not cause the intrinsic fluorescence quenching of W58 observed in the wild-type protein. The loop beta4-alpha4 appears to be very flexible in both mutants and helix alpha4, which starts at N94 in the native Mg2+-CheY and at K91 in the native apo-CheY, starts in both mutants at residue K92. The side-chain of K109 appears to be more mobile because of the space freed by the M17G mutation. In the triple mutant the main chain of K109 and adjacent residues (loop beta5-alpha5) is displaced almost by 2 angstrom affecting the main chain at residues T87 to E89 (C terminus of beta4). The triple mutant structure has a Mg2+ bound at the active site, but although the Mg2+ coordination is similar to that of the native Mg2+-CheY, the structural consequences of the metal binding are quite different. It seems that the mutations have disrupted the mechanism of movement transmission observed in the native protein. We suggest that the side-chain of K109, packed between V86, A88 and M17 in the native protein, slides forwards and backwards upon activation and deactivation dragging the main chain at the loop beta5-alpha5 and triggering larger movements at the functional surface of the protein.

Bacterial Proteins↗
PubMed1996

Conformational analysis of peptides corresponding to beta-hairpins and a beta-sheet that represent the entire sequence of the alpha-spectrin SH3 domain.

In an attempt to identify potential folding initiation sites for a small, all beta-protein domain, we have examined the conformational preferences in aqueous solution of peptides that span the entire length of the alpha-spectrin SH3 domain, using proton nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Two of the peptides correspond to beta-hairpins (m6 and m8), one to the RT-loop (m4, which can be considered as a distorted beta-hairpin), one to a beta-hairpin created by joining the N and C-terminal strands via a small linker (m2) and the fifth one to a three-stranded antiparallel beta-sheet composed of beta-hairpins m6 and m8 (m68). To estimate the distorting effect of the aromatic side-chains of Trp41 and Trp42 on the CD and NMR spectra of peptides m6, m8 and m68, we have also analyzed a short, ten-residue random-coil peptide containing residues 39 to 44 (mC). The CD and NMR results indicate that none of the peptides populates to a large extent a particular secondary structure conformation. However, careful anlaysis of the NMR data reveals that peptides m6, m8 and m68 could adopt, to a small extent, native-like conformations, although in the case of peptide m68 there is also evidence of the presence of non-native helical conformations. Addition of 30% (v/v) 2,2,2-trifluoroethanol stabilizes the appearance of non-native helical populations in some small regions of peptides m2, m4, m8 and m68, while it induces a native-like conformation in peptide m6. Those fragments corresponding to the two real beta-hairpins in the protein are the ones which exhibit some tendency to populate native-like structures (m6 and m8), while the ones corresponding to the long RT-loop (m4) or the newly created one (m2) are mainly unstructured in water solution. Although there could be some local interactions that favor the acquisition of a native secondary structure in this domain, tertiary interactions should play a major role in defining its native secondary structure.

Amino Acid Sequence↗
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

De novo design and structural analysis of a model beta-hairpin peptide system.

We have designed de novo a simple, context-free, model linear peptide system to fold into a regular beta-hairpin structure, with three-residue beta-strands connected by a type I' beta-turn. CD and NMR analysis of this peptide in aqueous solution show that the peptide folds into the expected conformation. Structural characterization of three peptide variants in which some of the strand side-chains have been substituted by alanine, demonstrates that inter-strand side chain-side chain interactions are essential for beta-hairpin formation. This simple model system will help to isolate the factors behind beta-sheet formation, and contribute useful information about de novo protein design.

Amino Acid Sequence↗
PubMed1996

Different folding transition states may result in the same native structure.

The crystal structures of two circular permutants of the alpha-spectrin SH3 domain with new termini within the RT loop (S19-P20s) and the distal loop (N47-D48s) have been determined at 2.02 and 1.77 A resolution respectively. Both fold into the same three-dimensional structure as the wild-type SH3 domain except for the engineered loop that fuses the wild-type termini. The cleaved RT loop in S19-P20s loses nine conserved hydrogen bonds through local hydrogen bond unzipping; no hydrogen bond unzipping occurs in N47-D48s. The structures of the transition states for folding of wild-type alpha-spectrin SH3 domain and the two circular permutants have been examined by analysis of the folding kinetics of eight strategically distributed point mutants. Unlike the native structures, the transition states of the three proteins are considerably different, suggesting that there is no direct relationship between these two states in a protein.

Amino Acid Sequence↗
PubMed1996

Stabilization of proteins by rational design of alpha-helix stability using helix/coil transition theory.

BACKGROUND: Increasing protein stability is a major goal of protein engineering because of its potential industrial and pharmacological applications. Several different rule-of-thumb strategies have been employed for such a purpose, but a general rational method is still lacking. Recently, there has been significant progress in our understanding of the interactions responsible for helix stability in monomeric peptides and this information has been included in algorithms based on the helix/coil transition theory. We set out to investigate whether it is possible to use these algorithms to rationally increase protein stability. RESULTS: Using a helix/coil transition algorithm, AGADIRms, we have designed mutations affecting solvent-exposed residues which, as predicted, significantly increase the helical stability in aqueous solution of peptides corresponding to the two alpha-helices of the activation domain of procarboxipeptidase A. Introduction of the same mutations in the protein results in proteins more resistant to urea or temperature denaturation, and there is a qualitative agreement between the expected and observed increases in stability. CONCLUSIONS: In this work we demonstrate that by using a helix/coil algorithm to design helix-stabilizing mutations on the solvent-exposed face of helices, it is possible to rationally increase the stability of proteins.

Algorithms↗
PubMed1996

Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2.

BACKGROUND: Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case. RESULTS: The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds. CONCLUSIONS: Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.

Amino Acid Sequence↗
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↗