Biomedical subjects

A R Viguera

Publications and source records attributed to A R Viguera.

At least 19 recordsLinked to original sources

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

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

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↗

PubMed1998

Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters.

The information about the conformational behavior of monomeric helical peptides in solution, as well as the alpha-helix stability in proteins, has been previously utilized to derive a database with the energy contributions for various interactions taking place in an alpha-helix: intrinsic helical propensities, side-chain-side-chain interactions, main-chain-main-chain hydrogen bonds, and capping effects. This database was implemented in an algorithm based on the helix/coil transition theory (AGADIR). Here, we have modified this algorithm to include previously described local motifs: hydrophobic staple, Schellman motif and Pro-capping motif, new variants of these, and newly described side-chain-side-chain interactions. Based on recent experimental data we have introduced a position dependence of the helical propensities for some of the 20 amino acid residues. A new electrostatic model that takes into consideration all electrostatic interactions up to 12 residues in distance in the helix and random-coil conformations, as well as the effect of ionic strength, has been implemented. We have synthesized and analyzed several peptides, and used data from peptides already analysed by other groups, to test the validity of our electrostatic model. The modified algorithm predicts, with an overall standard deviation value of 6.6 (maximum helix is 100%), the helical, content of 778 peptides of which 223 correspond to wild-type and modified protein fragments. To improve the prediction potential of the algorithm and to have a direct comparison with nuclear magnetic resonance data, the algorithm now predicts the conformational shift of the CalphaH protons, 13Calpha and 3JalphaN values. We have found that for those peptides correctly predicted from the point of view of circular dichroism, the prediction of the NMR parameters is very good.

Algorithms↗

PubMed1998

Reading protein sequences backwards.

BACKGROUND: Reading a protein sequence backwards provides a new polypeptide that does not align with its parent sequence. The foldability of this new sequence is questionable. On one hand, structure prediction at low resolution using lattice simulations for such a protein provided a model close to the native parent fold or to a topological mirror image of it. On the other hand, there is no experimental evidence yet to tell whether such a retro protein folds (and to which structure) or not. RESULTS: In this work, we have analysed the possibility of a retro protein folding in two different ways. First, we modelled the retro sequence of the alpha-spectrin SH3 domain through distance geometry and molecular dynamics. This contradicted the plausibility of a mirror image of the native domain, whereas basic considerations opposed the likelihood of the native fold. Second, we obtained experimental evidence that the retro sequences of the SH3 domain, as well as the B domain of Staphylococcal protein A and the B1 domain of Streptococcal protein G, are unfolded proteins, even though some propensities for the formation of secondary structures might remain. CONCLUSIONS: Retro proteins are no more similar to their parent sequences than any random sequence despite their common hydrophobic/hydrophilic pattern, global amino acid composition and possible tertiary contacts. Although simple folding models contribute to our global understanding of protein folding, they cannot yet be used to predict the structure of new proteins.

Amino Acid Sequence↗

PubMed1997

Favourable native-like helical local interactions can accelerate protein folding.

BACKGROUND: Extensive studies of peptide conformation have provided reasonable knowledge of the rules determining helix stability. This knowledge can be used to stabilize proteins against chemical and thermal denaturation. This has been done in two proteins: the chemotactic protein from Escherichia coli, Che Y (a 129 aa alpha/beta parallel protein with five alpha-helices, which shows an accumulating intermediate during refolding) and the activation domain of human procarboxypeptidase A2, ADA2h (a 81 aa alpha + beta protein domain, with two alpha-helices, which follows a two-state mechanism). As the introduced stabilizing interactions are local in nature, the energy balance between the contribution of local and nonlocal interactions changes considerably. Recent theoretical analyses of protein folding using simplified models have indicated that optimization of folding speed requires this balance to be biased towards nonlocal interactions. To determine whether this is the case, we study here the folding kinetics of two ADA2h mutants in which alpha-helix 1 (mutant M1) or 2 (mutant M2) has been stabilized through local interactions, as well as the equilibrium and kinetic behaviour of a double mutant (DM) in which both helices have been stabilized. RESULTS: The stability of DM is considerably enhanced with respect to wild type (WI) and this mutant can be considered as a thermoresistant protein (Tm > 363 K). The thermodynamic parameters obtained by chemical denaturation (urea and GdnHCl) show that DM is approximately 2.6 kcal mol-1 more stable than WT. The effects on folding kinetics are different in each of the single mutants. M1 shows very little effect in refolding, while its unfolding is greatly decelerated with respect to WT. M2 shows, together with a deceleration in unfolding, a significant acceleration in refolding. As with equilibrium parameters, the kinetics of the double mutant can be explained by the simple addition of the effects found in each single mutant. Interestingly enough, the refolding slope mkf in mutants M2 and DM is smaller than in the wild-type and M1 mutant. CONCLUSIONS: Thermoresistance can be achieved, in some cases, by increasing favourable native local interactions. The balance between local and nonlocal interactions can be significantly changed in some proteins and still keep a cooperative unfolding transition similar to that of the wild type. The introduction of favourable local interactions by mutational redesign can also be used to increase the folding speed of certain proteins, showing that not all proteins in nature have been optimized for rapid folding, contrary to what has been theoretically indicated. This behaviour is probably also shared by other polypeptides with highly unstructured denatured states. All these phenomena have been shown experimentally in ADA2h by mutations that increase helix stability. However, the effects promoted for such an approach in proteins with residual structure and/or intermediates in the denatured ensemble could be different. This has been shown by experiments performed on CheY in which the cooperativity of the folding process was greatly affected.

Adaptor Proteins, Signal Transducing↗

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↗

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

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↗

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

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↗

PubMed1994

Early and delayed stages in the solubilization of purple membrane by a polyoxyethylenic surfactant.

The purpose of this paper is to explore the reasons by which purple membrane solubilization by detergents takes hours, or even days, to reach equilibrium, while most biomembranes are solubilized in a matter of seconds, or minutes. With that aim, changes in the purple membrane absorption spectrum produced by hydrogenated Triton X-100 under equilibrium conditions (24 h) have been compared to those caused by the same surfactant in the minute, second and sub-second time scale. It is found that the various processes that accompany, or lead to, solubilization are already detected, and even reach an apparent equilibrium, in the 10 s that follow detergent addition. No new phenomena are detected in the following minutes, or hours, that are relevant to the process under study. This leads to the conclusion that the long solubilization process consists of the repeated operation of simple phenomena that are relatively fast in themselves. A hypothesis is proposed according to which the tight crystalline organization of the purple membrane prevents the insertion of detergent monomers in the lipid bilayer; instead, the surfactant would bind the periphery of the patches, i.e., the hydrocarbon-water contact region, and solubilization would take place gradually, from the periphery towards the core of the membrane patches, at a progressively lower rate as the amounts of free detergent and detergent-binding sites are decreased by the previous solubilization steps.

Kinetics↗

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

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

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↗

PubMed1993

Time-resolved and equilibrium measurements of the effects of poly(ethylene glycol) on small unilamellar phospholipid vesicles.

The effects of poly(ethylene glycol) (PEG) on sonicated unilamellar vesicles made of phosphatidylcholine have been examined. Stopped-flow and equilibrium data are presented for vesicle aggregation, vesicle leakage, lipid mixing, and aqueous contents mixing. Vesicle aggregation is detected as a monoexponential increase in light scattering, for PEG concentrations between 5 and 10%. In the region between 10-15% and 23-27% PEG, under our experimental conditions, the increase in light scattering follows a more complex biexponential kinetics, and, under these conditions, vesicle aggregation is accompanied by lipid mixing, a combination of events denoted as "close apposition". Above 23-27% PEG, the increase in light scattering is accompanied by fast lipid mixing, and also mixing of aqueous contents, all this being indicative of vesicle fusion; in addition, leakage occurs under these circumstances. Fusion takes place at high PEG concentrations, as indicated above, without any dilution step. From a methodological point of view, the Tb/DPA assay is shown to be more appropriate than the ANTS/DPX method for leakage and fusion studies in the presence of PEG.

Energy Transfer↗