Biomedical subjects

L Serrano

Publications and source records attributed to L Serrano.

At least 127 records · Page 7Linked to original sources

PubMed1994

Characterization of the interaction of natural proline-rich peptides with five different SH3 domains.

The interaction of six different proline-rich peptides with five SH3 domains has been investigated by using spectroscopic techniques. These peptides correspond to natural sequences and have been implicated in the interaction of some SH3 domains with other proteins. We have determined the Kd values for all of the possible combinations between the peptides and the SH3 domains. Low specificity and low affinity (> 5 microM) are the most remarkable conclusions from these studies. None of the peptides tested here were found to bind with significant affinity to spectrin-SH3 or n-src-SH3. Abl-SH3 seems to be the most selective of the domains analyzed here, while Fyn-SH3 is the most promiscuous. CD and FTIR studies indicate that these peptides adopt to different extents a PPII-like structure in aqueous solution. However, analysis of the SH3 domain complexes with these peptides suggests that proline-rich peptides do not necessarily adopt an overall PPII structure over their entire length upon binding to the different SH3 domains.

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↗

PubMed1994

Magnesium binding to the bacterial chemotaxis protein CheY results in large conformational changes involving its functional surface.

The three-dimensional crystal structure of the bacterial chemotaxis protein CheY with the essential Mg2+ cation bound to the active site reveals large conformational changes caused by the metal binding. Displacements of up to 10 A are observed in several residues at the N terminus of alpha-helix 4 and in the preceding loop. One turn of this helix unwinds, and an Asn residue that was located inside the helix becomes the new N-cap. This supports the important role that N or C-cap residues play in alpha-helix stability. In addition the preceding beta-strand becomes elongated and a new beta-turn appears. The final effect is a significant modification of the surface relief of the protein in a region previously indicated, by genetic analysis, to be essential for CheY function. It is suggested that binding of a divalent cation to CheY could play a significant part in CheY activation and consequently in signal transduction in prokaryotes.

Bacterial Proteins↗

PubMed1994

Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding transition.

The folding and unfolding reactions of the SH3 domain of spectrin can be described by a two-state model. This domain is a beta-sheet barrel containing 62 amino acids. Equilibrium unfolding by urea, guanidine hydrochloride, and heat is completely reversible at pH values below 4.0. At higher pH values the unfolding is reversible as long as the protein concentration is below 1 mg/mL. The Gibbs energy of unfolding in the absence of denaturant, delta GH2O, at pH 3.5 and 298 K is calculated to be 12 kJ mol-1 for urea, chemical, and temperature denaturation. The stability of the protein does not change noticeably between pH 5.0 and 7.0 and is around 15.5 kJ mol-1. Since heat effects of unfolding are relatively small and, as a result, heat-induced melting occurs in a wide temperature range, the analysis of scanning calorimetry data was performed taking into account the temperature dependence of unfolding delta Cp. The free energy of unfolding obtained for this domain (delta GH2O = 14 +/- 2 kJ mol-1) was, within experimental error, similar to those obtained in this work by other techniques and with those reported in the literature for small globular proteins. Kinetics of unfolding and refolding at pH 3.5, followed both by fluorescence and by circular dichroism, provide evidence of the simplest folding mechanism consistent with the two-state approximation. A value for delta GH2O = 13 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Homology modeling of the Abl-SH3 domain.

A tertiary structure model of the Abl-SH3 domain is predicted by using homology modeling techniques coupled to molecular dynamics simulations. Two template proteins were used, Fyn-SH3 and Spc-SH3. The refined model was extensively checked for errors using criteria based on stereochemistry, packing, solvation free-energy, accessible surface areas, and contact analyses. The different checking methods do not totally agree, as each one evaluates a different characteristic of protein structures. Several zones of the protein are more susceptible to incorporating errors. These include residues 13, 15, 35, 39, 45, 46, 50, and 60. An interesting finding is that the measurement of the C alpha chirality correlated well with the rest of the criteria, suggesting that this parameter might be a good indicator of correct local conformation. Deviations of more than 4 degrees may be indicative of poor local structure.

Computer Simulation↗

PubMed1994

Intrinsic secondary structure propensities of the amino acids, using statistical phi-psi matrices: comparison with experimental scales.

Today there are several different experimental scales for the intrinsic alpha-helix as well as beta-strand propensities of the 20 amino acids obtained from the thermodynamic analysis of various model systems. These scales do not compare well with those extracted from statistical analysis of three-dimensional structure databases. Possible explanations for this could be the limited size of the databases used, the definitions of intrinsic propensities, or the theoretical approach. Here we report a statistical determination of alpha-helix and beta-strand propensities derived from the analysis of a database of 279 three-dimensional structures. Contrary to what has been generally done, we have considered a particular residue as in alpha-helix or beta-strand conformation by looking only at its dihedral angles (phi-psi matrices). Neither the identity nor the conformation of the surrounding residues in the amino acid sequence has been taken into consideration. Pseudoenergy empirical scales have been calculated from the statistical propensities. These scales agree very well with the experimental ones in relative and absolute terms. Moreover, its correlation with the average of the experimental scales for alpha-helix or beta-strand is as good as the correlations of the individual experimental scales with the average. These results show that by using a large enough database and a proper definition for the secondary structure propensities, it is possible to obtain a scale as good as any of experimental origin. Interestingly the phi-psi analysis of the Ramachandran plot suggests that the amino acids could have different beta-strand propensities in different subregions of the beta-strand area.

Amino Acids↗

PubMed1994

Elucidating the folding problem of helical peptides using empirical parameters.

Using an empirical analysis of experimental data we have estimated a set of energy contributions which accounts for the stability of isolated alpha-helices. With this database and an algorithm based on statistical mechanics, we describe the average helical behaviour in solution of 323 peptides and the helicity per residue of those peptides analyzed by nuclear magnetic resonance. Moreover the algorithm successfully detects the alpha-helical tendency, in solution, of a peptide corresponding to a beta-strand of ubiquitin.

Algorithms↗

PubMed1994

A short linear peptide that folds into a native stable beta-hairpin in aqueous solution.

The conformational properties of a 16 residue peptide, corresponding to the second beta-hairpin of the B1 domain of protein G, have been studied by nuclear magnetic resonance spectroscopy (NMR). This fragment is monomeric under our experimental conditions and in pure water adopts a population containing up to 40% native-like beta-hairpin structure. The detection by NMR of a native-like beta-hairpin in aqueous solution, reported here for the first time, indicates that these structural elements may have an important role in the early steps of protein folding. It also provides a good model to study in detail the sequence determinants of beta-hairpin structure stability, as has been done with alpha-helices.

Amino Acid Sequence↗

PubMed1994

Molecular modeling of the interaction of polyproline-based peptides with the Abl-SH3 domain: rational modification of the interaction.

A molecular model of the interaction of polyproline-rich peptides with the Abl-SH3 domain is proposed, based on docking calculations with the DOCK program coupled with molecular dynamics simulations. Two distinct binding modes of the peptide to the same aromatic-rich region (Tyr10, Phe12, Trp39, Trp50, Tyr55) of the domain were obtained. It is proposed that these two models could represent different binding modes of proline-rich peptides to Src homology region 3 domains. Several peptide mutants were designed to determine whether the two orientations were possible. Analysis of the Kd values and fluorescence emission of these peptides indicate that one of the orientations is more plausible and that residues at position 4 of the peptide interact with the RT loop, being important in modulating the peptide affinity for the Abl-SH3 domain.

Amino Acid Sequence↗

PubMed1994

Albert's solution versus potassium hydroxide solution in the diagnosis of tinea versicolor.

BACKGROUND: The diagnosis of tinea versicolor can usually be made by clinical examination. Either potassium hydroxide (KOH) or Wood's light fluorescence are used to corroborate the diagnosis. Albert's solution has been reported as a method of visualizing dermatophytes. We compared Albert's solution to KOH in the diagnostic evaluation of tinea versicolor. METHODS: Twenty patients with clinical tinea versicolor were examined for the presence of hyphae and/or spores utilizing Albert's solution or KOH (20%) with dimethyl sulfoxide. RESULTS: All patients were positive for hyphae and/or spores by both methods. Preparations with Alberts' stains were faster and easier to read. CONCLUSIONS: Albert's solution is an alternate method to stain for fungal elements in patients with tinea versicolor.

Acetates↗

PubMed1994

[Laparoscopic gastrostomy].

Feeding gastrostomy is a method for treating patients unable to ingest food orally. After our experience and good results with the Janeway technique, we decided to perform it by the laparoscopic method. Our first excellent results in seven patients suggest that laparoscopic Janeway gastrostomy is an easy, rapid and efficient surgical technique.

Aged↗

PubMed1993

Thermodynamic analysis of the chemotactic protein from Escherichia coli, CheY.

CheY, the 129 amino acid chemotactic protein from Escherichia coli, is a good model for studies of folding of parallel alpha/beta proteins. We report here the thermodynamic characterization of the wild-type CheY at different pH values and in different buffers and denaturation conditions. The denaturation of CheY by urea monitored by circular dichroism and fluorescence fits the two-state unfolding model. The stability of the protein is ionic strength dependent, probably due to the presence of three Asp residues in very close proximity in its active site. The presence of a Mg2+ ion, which seems to interact with Asp 13 in the active site, stabilizes the native structure by up to 6.9 kJ mol-1. The CheY maximum stability (31.7 +/- 2.1 kJ mol-1), without magnesium, is reached at pH 5.1. Analysis of scanning calorimetry data has shown that temperature-induced unfolding of CheY is not a two-state process and proceeds through a highly populated intermediate state, corresponding to protein dimers, as was subsequently confirmed by direct cross-linking experiments. According to circular dichroism, fluorescence, nuclear magnetic resonance, and ANS binding experiments, this "intermediate dimer" at pH 2.5 exhibits all known characteristics of the "molten globule" state. The reversible dimerization of "molten globules" might explain such peculiarities as the increased stability or the cooperative unfolding found for the molten globule state of some proteins.

Anilino Naphthalenesulfonates↗

PubMed1993

Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability.

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant.

Amino Acid Sequence↗

PubMed1993

1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein.

Che Y is a 129-residue parallel alpha/beta protein involved in bacterial chemotaxis. We have used this protein as a model to study the folding reaction of parallel alpha/beta proteins. As a first step we carried out the complete assignment of the 1H and 15N spectra from Escherichia coli Che Y protein on the basis of two-dimensional 1H homonuclear and 1H-15N heteronuclear experiments by using sequence-specific methods. Our assignments differ from the preliminary assignments made by Kar et al. [Kar, L., Matsumura, P. & Johnson, M.E. (1992) Biochem. J. 287, 521-531] of aromatic residues obtained by comparison of NOEs with short proton-proton distances in the crystal structure of Che Y. The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three-dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X-ray diffraction.

Amino Acid Sequence↗

PubMed1993

Role of the carboxy terminal region of beta tubulin on microtubule dynamics through its interaction with the GTP phosphate binding region.

The dynamic instability of microtubules depends on the GTP binding to tubulin, the rate of hydrolysis of GTP bound to tubulin molecules, at the microtubule caps, and on the affinity and exchange rate of tubulin for GTP versus GDP. It has been demonstrated that the binding of microtubule-associated proteins (MAPs) such as Tau or MAP2 notably enhances microtubule stability in vivo. These MAPs bind to the tubulin carboxy terminal domain. Consequently, an attractive hypothesis to explain the modulation of microtubule dynamics by MAPs is that the carboxy terminal domain of tubulin interacts with a region close to the GTP binding site, preventing the binding of GTP or exchange of GDP for GTP. By carrying out a combined analysis of crosslinking and limited proteolysis, an intramolecular interaction between the carboxy terminus and the tubulin region containing the GTP binding site in beta tubulin has been observed. It is proposed that this interaction modifies the binding of GTP to the tubulin beta-subunit and, therefore, affects tubulin assembly dynamics. This suggests a molecular explanation for the effect of MAPs in facilitating tubulin polymerization through the regulation of the interaction of GTP.

Amino Acid Sequence↗

PubMed1992

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗

PubMed1992

Effect of active site residues in barnase on activity and stability.

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.

Bacillus↗

PubMed1992

The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding.

The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter phi. There are two extreme values of phi that are often found in practice and may be interpreted in a simple manner. A value of phi = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, phi = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of phi are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of phi found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of phi = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the double-mutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.

Bacterial Proteins↗