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D Perahia

Publications and source records attributed to D Perahia.

At least 19 recordsLinked to original sources
PubMed2000

Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface.

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.

Animals↗
PubMed1999

Tertiary and quaternary conformational changes in aspartate transcarbamylase: a normal mode study.

Aspartate transcarbamylase (ATCase) initiates the pyrimidine biosynthetic pathway in Escherichia coli. Binding of aspartate to this allosteric enzyme induces a cooperative transition between the tensed (T) and relaxed (R) states of the enzyme which involves large quaternary and tertiary rearrangements. The mechanisms of the transmission of the regulatory signal to the active site (60 A away) and that of the cooperative transition are not known in detail, although a large number of single, double, and triple site-specific mutants and chimeric forms of ATCase have been obtained and kinetically characterized. A previous analysis of the very low-frequency normal modes of both the T and R state structures of ATCase identified some of the large-amplitude motions mediating the intertrimer elongation and rotation that occur during the cooperative transition (Thomas et al., J. Mol. Biol. 257:1070-1087, 1996; Thomas et al., J. Mol. Biol. 261:490-506, 1996). As a complement to that study, the deformation of the quaternary and tertiary structure of ATCase by normal modes below 5 cm(-1) is investigated in this article. The ability of the modes to reproduce the domain motions occurring during the transition is analyzed, with special attention to the interdomain closure in the catalytic chain, which has been shown to be critical for homotropic cooperativity. The calculations show a coupling between the quaternary motions and more localized motions involving specific residues. The particular dynamic behavior of these residues is examined in the light of biochemical results to obtain insights into their role in the transmission of the allosteric signal.

Allosteric Site↗
PubMed1998

1.2 A refinement of the Kunitz-type domain from the alpha3 chain of human type VI collagen.

The recombinant Kunitz-type domain (C5) of human collagen alpha3(VI) chain was previously described at 1.6 A resolution at room temperature. By changing the crystallization conditions and using synchrotron radiation, we are able to record diffraction data to 1.2 A resolution for crystals of the same space group at 291 K. The protein-water-ion model has been refined anisotropically against these new data using the program SHELXL93; the results converged to an R factor of 15.0%, with all data between 7 and 1.2 A. The final electron-density map reveals a clear chain tracing with a few disordered residues and five residues out of 58 that present alternate conformations. The Cys14-Cys38 bond presents the less frequently observed left-hand conformation (chi1 = -60 degrees). The solvent molecules and a phosphate ion are well ordered with an average B of 38 A2. The high-resolution structure reveals the N and C termini which were missing from the 1.6 A structure.

Amino Acid Sequence↗
PubMed1998

Conformational dynamics and enzyme activity.

Conformational flexibility and structural fluctuations play an important role in enzyme activity. A great variety of internal motions ranging over different time scales and of different amplitudes are involved in the catalytic cycle. These different types of motions and their functional consequences are considered in the light of experimental data and theoretical analyses. The conformational changes upon substrate binding, and particularly the hinge-bending motion which occurs in enzymes made of two domains, are analyzed from several well documented examples. The conformational events accompanying the different steps of the catalytic cycle are discussed. The last section concerns the motions involved in the allosteric transition which regulates the enzyme activity.

Allosteric Regulation↗
PubMed1996

Analysis of the low-frequency normal modes of the R state of aspartate transcarbamylase and a comparison with the T state modes.

Aspartate transcarbamylase (ATCase) is a classic example of an allosteric enzyme. It catalyzes the conversion of aspartate to carbamyl aspartate, which is the first substrate in the biosynthesis of pyrimidines. Although ATCase is well characterized, both structurally and biochemically, little is known at the atomic level about the large amplitude motions that govern its T-->R quaternary transition. We present the results of calculations of the very-low-frequency normal modes of the CTP-ligated R state ATCase, and we compare them with the equivalent modes in the CTP-ligated T state ATCase. The large-amplitude, delocalized modes of frequencies below 4 cm-1 contribute a large fraction of the atomic fluctuations observed experimentally. They show some ability to drive the R-state structure towards the T-state structure, by promoting some of the quaternary structure rearrangements that take place during the allosteric process. Their potential role in the T-->R transition is quantified and compared with the role of the low-frequency modes of the T state in the quaternary rearrangement.

Allosteric Regulation↗
PubMed1996

Motions in hemoglobin studied by normal mode analysis and energy minimization: evidence for the existence of tertiary T-like, quaternary R-like intermediate structures.

The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like.

Heme↗
PubMed1996

Analysis of the low frequency normal modes of the T-state of aspartate transcarbamylase.

Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed.

Allosteric Site↗
PubMed1996

Polar fluctuations in proteins: molecular-dynamic studies of cytochrome c in aqueous solution.

The equilibrium fluctuations (the polar fluctuations), of yeast cytochrome c are studied using nanosecond molecular-dynamic simulations in a spherical droplet of water, with a particular emphasis on the fluctuations of the total dipole moment, which determine the average relative permittivity. These fluctuations follow a simple probability distribution, predicted by continuum electrostatics, and already observed in simulations of several polar liquids. An important component consists of diffusive, mutually independent, motions of the charged side chains at the protein surface. A quasiharmonic normal mode analysis of the trajectory shows that while motions covering a large range of frequencies contribute to the polar fluctuations, the four lowest frequency modes account for 50% of the overall static relative permittivity of ca. 25. The fluctuations of the protein bulk, i.e. parts other than the charged side chains, are distributed over a larger number of modes. Modes up to at least 60 cm-1 contribute to the average relative permittivity of the protein interior of ca. 4. The water surrounding the protein, despite the structural perturbation represented by the protein, has fluctuations similar to pure water, consistent with the idea of a linear solvent response to the protein charges. The relationship between the microscopic fluctuations seen in the simulations and simple continuum models is discussed.

Cytochrome c Group↗
PubMed1995

Internal and interfacial dielectric properties of cytochrome c from molecular dynamics in aqueous solution.

The dielectric properties of proteins are central to their stability and activity. We use the Fröhlich-Kirkwood theory of dielectrics to analyze two 1-ns molecular dynamics simulations of ferro- and ferricytochrome c in spherical droplets of 1400 water molecules. Protein and solvent are idealized as a series of concentric, spherical, dielectric media. Analysis results depend strongly on the treatment of the charged protein side chains at the protein/solvent interface. If charged side chains are viewed as part of the protein medium, then the protein dipole fluctuations are dominated by large, mutually uncorrelated, anisotropic, motions of the charged side chains. It is then incorrect to view the protein region as a single, homogeneous dielectric material. If one does take this view, estimates of the protein "dielectric constant" vary from 16 to 37, depending on the exact choice of model parameters. In contrast, if the charged portions of the charged side chains are viewed as part of the solvent medium, then theory and simulation are consistent: the protein dipole fluctuations excluding charged side chains are roughly those of a homogeneous, isotropic dielectric medium, with a dielectric constant of 4.7 +/- 1.0 (ferro) or 3.4 +/- 1.0 (ferri), in agreement with powder experiments. Statistical uncertainty and sensitivity to model parameters are small. Analysis of the radial dependence of the dipole fluctuations suggests that the inner half of the protein has a somewhat lower dielectric constant of 1.5-2, consistent with its biological function in electron transfer. These results suggest that Poisson-Boltzmann models could treat the protein bulk as a low-dielectric medium and the charged surface groups as part of the solvent region.

Cytochrome c Group↗
PubMed1995

Computation of low-frequency normal modes in macromolecules: improvements to the method of diagonalization in a mixed basis and application to hemoglobin.

The method of diagonalization in a mixed basis (DIMB) that was published previously (Mouawad.), L. and Perahia D., Biopolymers 33, 599, 1993), allows the computation of the low-frequency vibrational modes for large macromolecules. Improvements to this method are presented here, namely the single and double truncation window techniques. The best convergence rate is obtained with the double truncation windows, which couple most efficiently the parts of the macromolecule which are far in sequence but close in space. Both methods were applied to the T-state of hemoglobin, to compare their efficiency. The resulting modes are analyzed in order to study the pathways between T- and R-states of this protein. They show that the quaternary conformational are mainly due to one mode at 2 cm-1.

Hemoglobins↗
PubMed1994

A free-energy simulation study of a bacterial collagenase inhibitor.

The cis-trans conformational isomerisation of the N-methylated peptide bond of a bacterial collagenases peptide inhibitor (HS-CH2-CH2-CO-Pro-NMe-Ala) has been investigated by molecular dynamics simulations, energy minimisations and free-energy simulations in presence of solvent molecules. The free-energy difference between the cis and trans forms obtained by the thermodynamics integration method is equal to 0.95 kcal/mol in favour of the trans form, in accord with the experimental result. The main contribution to this free-energy difference comes from solute-water electrostatic interactions. Interestingly, we show that the number of interactions between water molecules and the oxygen atoms of the inhibitor is larger in the trans form than in the cis. Thus the organisation of water molecules around the inhibitor appears crucial in determining the population of the cis and trans conformers.

Bacteria↗
PubMed1992

Normal modes of symmetric protein assemblies. Application to the tobacco mosaic virus protein disk.

We use group theoretical methods to study the molecular dynamics of symmetric protein multimers in the harmonic or quasiharmonic approximation. The method explicitly includes the long-range correlations between protein subunits. It can thus address collective dynamic effects, such as cooperativity between subunits. The n lowest-frequency normal modes of each individual subunit are combined into symmetry coordinates for the entire multimer. The Hessian of the potential energy is thereby reduced to a series of blocks of order n or 2n. In the quasiharmonic approximation, the covariance matrix of the atomic oscillations is reduced to the same block structure by an analogous set of symmetry coordinates. The method is applied to one layer of the tobacco mosaic virus protein disk in vacuo, to gain insight into the role of conformational fluctuations and electrostatics in tobacco mosaic virus assembly. The system has 78,000 classical, positional, degrees of freedom, yet the calculation is reduced by symmetry to a problem of order 4,600. Normal modes in the 0-100 cm-1 range were calculated. The calculated correlations extend mainly from each subunit to its nearest neighbors. The network of core helices has weak correlations with the rest of the structure. Similarly, the inner loops 90-108 are uncorrelated with the rest of the structure. Thus, the model predicts that the dielectric response in the RNA-binding region is mainly due to the loops alone.

Binding Sites↗
PubMed1991

Intramolecular dielectric screening in proteins.

This paper investigates the microscopic mechanisms of charge screening by proteins. For this purpose, we introduce the generalized susceptibility of a protein in response to a point charge, which is a scalar quantity dependent on position within the protein. The contribution to the susceptibility from atomic polarizabilities, associated with electronic degrees of freedom, is found to be highly uniform. By contrast, that from dynamic dipolar relaxation, associated with nuclear degrees of freedom, varies greatly between different regions of the protein. We investigate the possible rôle of this variation in the activity of proteins that interact functionally with charged species, and we formulate and test the hypothesis that this variation is correlated to functional activity. Model calculations give encouraging support to this hypothesis. The protein's dielectric properties are represented by a standard model in which electronic relaxation is described by a set of atomic polarizabilities, and dipolar relaxation is treated as a perturbation to normal mode dynamics. The model yields the desired susceptibility in closed form. Its obvious limitations are discussed. It is applied to several test systems, and is compared to various continuum models. Four model alpha-helices are considered, three of which play a rôle in vivo in the binding of charged ligands. We show that the intramolecular screening, and its spatial variation, can indeed play a part in this binding. The electron transfer between ferri- and ferrocytochrome c is considered. The dielectric relaxation of each molecule, associated respectively with its oxidation or its reduction, is known to be directly related to the activation free energy for the electron transfer reaction. Our analysis of the dielectric susceptibility will thus permit an estimate of this activation free energy. We show that the relaxation of the atomic positions ("dipolar relaxation") contributes 1 kcal/mol to this activation free energy, and that the molecule achieves this low value by providing a low dipolar susceptibility throughout its central part. In this case, the spatial variation of the susceptibility has a clear functional rôle.

Amino Acid Sequence↗
PubMed1991

Microscopic theory of the dielectric properties of proteins.

This paper investigates the microscopic mechanisms of charge screening in proteins. The screening of an arbitrary perturbing charge density by a protein and its surrounding solution is characterized by a generalized susceptibility, which is approximately given by the mean dipole-dipole correlation matrix of the system. This susceptibility is a microscopic quantity; the sum of its matrix elements gives the macroscopic susceptibility of continuum electrostatics. When screening of a single perturbing point charge is considered, this susceptibility reduces to a scalar quantity, dependent on position within the protein. The contribution of the positional degrees of freedom of the protein atoms can be estimated from molecular dynamics simulations. This contribution gives rise to large spatial variations of the susceptibility, whose significance for protein function is discussed. The model is applied to the small alpha helix deca-alanine, and to the electron-transfer protein cytochrome c. The results agree qualitatively with previous normal mode calculations. The importance, and the large spatial variations, of charge screening by deca-alanine suggest that dielectric screening may play a role in the binding of charged ligands by helices. In cytochrome c, the dielectric susceptibility in response to a point charge is at a minimum in the central heme region, resulting in a lowering of the reorganization free energy for charge transfer to and from the heme.

Biophysical Phenomena↗
PubMed1990

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation.

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.

Adenosine Diphosphate↗
PubMed1990

Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase.

Several mutants of yeast phosphoglycerate kinase, each containing only one internal cysteine residue, were constructed from a single mutant devoid of cysteine. These cysteines were introduced as local conformational probes in selected buried positions. The enzyme activity, conformational characteristics and stability indicated that the mutations introduced only small perturbations in the molecule. The folding-unfolding process mediated by guanidine hydrochloride under equilibrium conditions was studied by following the variations in ellipticity and the reactivity of the cysteine residue towards 5,5'-dithiobis(nitrobenzoate). The process was found to be reversible except for mutant C97A, V49C, suggesting that this region located in helix I might be crucial in determining an intermediate on the folding pathway. The transitions obtained by the two signals did not coincide, indicating that the local structures, in several parts inside the molecule, are more sensitive to the denaturant than the overall conformation.

Catalysis↗
PubMed1989

Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis.

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.

Animals↗