Biomedical subjects

A Caflisch

Publications and source records attributed to A Caflisch.

At least 19 recordsLinked to original sources

PubMed2006

A molecular dynamics approach to the structural characterization of amyloid aggregation.

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.

Amino Acid Sequence↗

PubMed2004

Replica exchange molecular dynamics simulations of amyloid peptide aggregation.

The replica exchange molecular dynamics (REMD) approach is applied to four oligomeric peptide systems. At physiologically relevant temperature values REMD samples conformation space and aggregation transitions more efficiently than constant temperature molecular dynamics (CTMD). During the aggregation process the energetic and structural properties are essentially the same in REMD and CTMD. A condensation stage toward disordered aggregates precedes the beta-sheet formation. Two order parameters, borrowed from anisotropic fluid analysis, are used to monitor the aggregation process. The order parameters do not depend on the peptide sequence and length and therefore allow to compare the amyloidogenic propensity of different peptides

Amyloid↗

PubMed2004

Formation of the folding nucleus of an SH3 domain investigated by loosely coupled molecular dynamics simulations.

The experimentally well-established folding mechanism of the src-SH3 domain, and in particular the phi-value analysis of its transition state, represents a sort of testing table for computational investigations of protein folding. Here, parallel molecular dynamics simulations of the src-SH3 domain have been performed starting from denatured conformations. By rescuing and restarting only trajectories approaching the folding transition state, an ensemble of conformations was obtained with a completely structured central beta-sheet and a native-like packing of residues Ile-110, Ala-121, and Ile-132. An analysis of the trajectories shows that there are several pathways leading to the formation of the central beta-sheet whereas its two hairpins form in a different but consistent way.

Computer Simulation↗

PubMed2001

Forces and energetics of hapten-antibody dissociation: a biased molecular dynamics simulation study.

The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-defined pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the fluorescein exit from the binding site. This occurs in steps that involve breaking of specific electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in significant changes in the unbinding force.

Animals↗

PubMed2001

Role of native topology investigated by multiple unfolding simulations of four SH3 domains.

The relative importance of amino acid sequence and native topology in the unfolding process of two SH3 domains and two circular permutants was investigated by 120 molecular dynamics runs at 375 K for a total simulation time of 0.72 micros. The alpha-spectrin (aSH3) and src SH3 (sSH3) domains, which have the same topology and a sequence identity of only 34%, show similar unfolding pathways. The disappearance of the three-stranded antiparallel beta-sheet is the last unfolding event, in agreement with a large repertoire of kinetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of beta-sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferred in aSH3 (n-src loop breaks before distal hair-pin) and the inverse in sSH3. An elongation of the beta2-beta3 hairpin was observed during the unfolding of sSH3 at 375 K and in 300 K simulations started from the putative transition state of sSH3 in accord with unusual kinetic data for point mutations at the n-src loop. The change of connectivity in the permutants influenced the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of contact disappearance. Taken together with previous folding simulations of two designed three-stranded antiparallel beta-sheet peptides, these results indicate that, at least for small beta-sheet proteins, the folding mechanism is primarily defined by the native state topology, whilst specific interactions determine the statistically predominant folding route.

Amino Acid Sequence↗

PubMed2001

Native topology or specific interactions: what is more important for protein folding?

Fifty-five molecular dynamics runs of two three-stranded antiparallel beta-sheet peptides were performed to investigate the relative importance of amino acid sequence and native topology. The two peptides consist of 20 residues each and have a sequence identity of 15 %. One peptide has Gly-Ser (GS) at both turns, while the other has d-Pro-Gly ((D)PG). The simulations successfully reproduce the NMR solution conformations, irrespective of the starting structure. The large number of folding events sampled along the trajectories at 360 K (total simulation time of about 5 micros) yield a projection of the free-energy landscape onto two significant progress variables. The two peptides have compact denatured states, similar free-energy surfaces, and folding pathways that involve the formation of a beta-hairpin followed by consolidation of the unstructured strand. For the GS peptide, there are 33 folding events that start by the formation of the 2-3 beta-hairpin and 17 with first the 1-2 beta-hairpin. For the (D)PG peptide, the statistical predominance is opposite, 16 and 47 folding events start from the 2-3 beta-hairpin and the 1-2 beta-hairpin, respectively. These simulation results indicate that the overall shape of the free-energy surface is defined primarily by the native-state topology, in agreement with an ever-increasing amount of experimental and theoretical evidence, while the amino acid sequence determines the statistically predominant order of the events.

Computer Simulation↗

PubMed2001

Efficient electrostatic solvation model for protein-fragment docking.

A method is presented for the fast evaluation of the binding energy of a protein-small molecule complex with electrostatic solvation. It makes use of a fast preprocessing step based on the assumption that the main contribution to electrostatic desolvation upon ligand binding originates from the displacement of the first shell of water molecules. For a rigid protein, the precomputation of the energy contributions on a set of grids allows the estimation of the energy in solution of about 300 protein-fragment binding modes per second on a personal computer. The docking procedure is applied to five rigid binding sites whose size ranges from 17 residues to a whole protein of 107 amino acids. Using a library of 70 mainly rigid molecules, known micromolar inhibitors or close analogs are docked and prioritized correctly. The docking based rank-ordering of the library requires about 5 h and is proposed as a complementary approach to structure-activity relationships by nuclear magnetic resonance. Proteins 2001;42:256-268.

Binding Sites↗

PubMed2001

Hydrophobicity and functionality maps of farnesyltransferase.

Farnesyltransferase (FTase) catalyzes the attachment of a 15-carbon isoprenoid moiety, farnesyl, through a thioether linkage to a cysteine near the C-terminus of oncogenic Ras proteins. These transform animal cells to a malignant phenotype when farnesylated. Hence, FTase is an interesting target for the development of antitumor agents. In this work we first investigate the active site of FTase by mapping its hydrophobic patches. Then the program SEED is used to dock functional groups into the active site by an exhaustive search and efficient evaluation of the binding energy with solvation. The electrostatic energy is SEED is based on the continuum dielectric approximation and consists of screened intermolecular energy and protein and fragment desolvation terms. The results are found to be consistent with the sequence variability of the tetrapeptide substrate. The distribution of functional groups (functionality maps) on the substrate binding site allows for identification of modifications of the tetrapeptide sequence that are consistent with potent peptidic inhibitors. Furthermore, the best minima of benzene match corresponding moieties of an inhibitor in clinical trials. The functionality maps are also used to design a library of disubstituted indoles that might prevent the binding of the protein substrates.

Alkyl and Aryl Transferases↗

PubMed2001

Flexibility of the murine prion protein and its Asp178Asn mutant investigated by molecular dynamics simulations.

Inherited forms of transmissible spongiform encephalopathy, e.g. familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia, segregate with specific point mutations of the prion protein. It has been proposed that the pathologically relevant Asp178Asn (D178N) mutation might destabilize the structure of the prion protein because of the loss of the Arg164-Asp178 salt bridge. Molecular dynamics simulations of the structured C-terminal domain of the murine prion protein and the D178N mutant were performed to investigate this hypothesis. The D178N mutant did not deviate from the NMR conformation more than the wild type on the nanosecond time scale of the simulations. In agreement with CD spectroscopy experiments, no major structural rearrangement could be observed for the D178N mutant, apart from the N-terminal elongation of helix 2. The region of structure around the disulfide bridge deviated the least from the NMR conformation and showed the smallest fluctuations in all simulations in agreement with hydrogen exchange data of the wild type prion protein. Large deviations and flexibility were observed in the segments which are ill-defined in the NMR conformation. Moreover, helix 1 showed an increased degree of mobility, especially at its N-terminal region. The dynamic behavior of the D178N mutant and its minor deviation from the folded conformation suggest that the salt bridge between Arg164 and Asp178 might not be crucial for the stability of the prion protein.

Animals↗

PubMed2001

Fragment-Based flexible ligand docking by evolutionary optimization.

A new computational approach for the efficient docking of flexible ligands in a rigid protein is presented. It exploits the binding modes of functional groups determined by an exhaustive search with solvation. The search in ligand conformational space is performed by a genetic algorithm whose scoring function approximates steric effects and intermolecular hydrogen bonds. Ligand conformations generated by the genetic algorithm are docked in the protein binding site by optimizing the fit of their fragments to optimal positions of chemically related functional groups. We show that the use of optimal binding modes of molecular fragments allows to dock known inhibitors with about ten rotatable bonds in the active site of the uncomplexed and complexed conformations of thrombin and HIV-1 protease.

Algorithms↗

PubMed2001

An evolutionary approach for structure-based design of natural and non-natural peptidic ligands.

A new computational approach (PEP) is presented for the structure-based design of linear polymeric ligands consisting of any type of amino acid. Ligands are grown from a seed by iteratively adding amino acids to the growing construct. The search in chemical space is performed by a build-up approach which employs all of the residues of a user-defined library. At every growing step, a genetic algorithm is used for conformational optimization of the last added monomer inside the binding site of a rigid target protein. The binding energy with electrostatic solvation is evaluated to select sequences for further growing. PEP is tested on the peptide substrate binding site of the insulin receptor tyrosine kinase and farnesyltransferase. In both test cases, the peptides designed by PEP correspond to the sequence motifs of known substrates. For tyrosine kinase, tyrosine residues are suggested at position P and P+2. While the tyrosine at P is in agreement with the experimental data, the one at P+2 is a prediction which awaits experimental validation. For farnesyltransferase, it is shown that electrostatic solvation is necessary for the correct design of peptidic inhibitors.

Algorithms↗

PubMed2000

Folding simulations of a three-stranded antiparallel beta -sheet peptide.

Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sampled during 47 molecular dynamics trajectories for a total simulation time of more than 4 micros provide an atomic detail picture of the folding of a 20-residue synthetic peptide with a stable three-stranded antiparallel beta-sheet fold. The simulations successfully reproduce the NMR solution conformation, irrespective of the starting structure. The sampling of the conformational space is sufficient to determine the free energy surface and localize the minima and transition states. The statistically predominant folding pathway involves the formation of contacts between strands 2 and 3, starting with the side chains close to the turn, followed by association of the N-terminal strand onto the preformed 2-3 beta-hairpin. The folding mechanism presented here, formation of a beta-hairpin followed by consolidation, is in agreement with a computational study of the free energy surface of another synthetic three-stranded antiparallel beta-sheet by Bursulaya and Brooks [(1999) J. Am. Chem. Soc. 121, 9947-9951]. Hence, it might hold in general for antiparallel beta-sheets with short turns.

Models, Molecular↗

PubMed2000

Computer simulations of protein folding by targeted molecular dynamics.

We have performed 128 folding and 45 unfolding molecular dynamics runs of chymotrypsin inhibitor 2 (CI2) with an implicit solvation model for a total simulation time of 0.4 microseconds. Folding requires that the three-dimensional structure of the native state is known. It was simulated at 300 K by supplementing the force field with a harmonic restraint which acts on the root-mean-square deviation and allows to decrease the distance to the target conformation. High temperature and/or the harmonic restraint were used to induce unfolding. Of the 62 folding simulations started from random conformations, 31 reached the native structure, while the success rate was 83% for the 66 trajectories which began from conformations unfolded by high-temperature dynamics. A funnel-like energy landscape is observed for unfolding at 475 K, while the unfolding runs at 300 K and 375 K as well as most of the folding trajectories have an almost flat energy landscape for conformations with less than about 50% of native contacts formed. The sequence of events, i.e., secondary and tertiary structure formation, is similar in all folding and unfolding simulations, despite the diversity of the pathways. Previous unfolding simulations of CI2 performed with different force fields showed a similar sequence of events. These results suggest that the topology of the native state plays an important role in the folding process.

Computer Simulation↗

PubMed2000

Design of dimerization inhibitors of HIV-1 aspartic proteinase: a computer-based combinatorial approach.

Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.

Aspartic Acid Endopeptidases↗

PubMed1999

Hydrophobicity at the surface of proteins.

A new method is presented to quantitatively estimate and graphically display the propensity of nonpolar groups to bind at the surface of proteins. It is based on the calculation of the binding energy, i.e., van der Waals interaction plus protein electrostatic desolvation, of a nonpolar probe sphere rolled over the protein surface, and on the color coding of this quantity on a smooth molecular surface (hydrophobicity map). The method is validated on ten protein-ligand complexes and is shown to distinguish precisely where polar and nonpolar groups preferentially bind. Comparisons with existing approaches, like the display of the electrostatic potential or the curvature, illustrate the advantages and the better predictive power of the present method. Hydrophobicity maps will play an important role in the characterization of binding sites for the large number of proteins emerging from the genome projects and structure modeling approaches.

Alkyl and Aryl Transferases↗

PubMed1999

Exhaustive docking of molecular fragments with electrostatic solvation.

A new method is presented for docking molecular fragments to a rigid protein with evaluation of the binding energy. Polar fragments are docked with at least one hydrogen bond with the protein while apolar fragments are positioned in the hydrophobic pockets. The electrostatic contribution to the binding energy, which consists of screened intermolecular energy and protein and fragment desolvation terms, is evaluated efficiently by a numerical approach based on the continuum dielectric approximation. The latter is also used to predetermine the hydrophobic pockets of the protein by rolling a low dielectric sphere over the protein surface and calculating the electrostatic desolvation of the protein and van der Waals interaction energy. The method was implemented in the program SEED (solvation energy for exhaustive docking). The SEED continuum electrostatic approach has been successfully validated by a comparison with finite difference solutions of the Poisson equation for more than 2,500 complexes of small molecules with thrombin and the monomer of HIV-1 aspartic proteinase. The fragments docked by SEED in the active site of thrombin reproduce the structural features of the interaction patterns between known inhibitors and thrombin. Moreover, the combinatorial connection of these fragments yields a number of compounds that are very similar to potent inhibitors of thrombin. Proteins 1999;37:88-105.

Computer Simulation↗

PubMed1999

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.

Bacterial Proteins↗

PubMed1999

Solution conformation of phakellistatin 8 investigated by molecular dynamics simulations.

Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and structure similar to antamanide. In molecular dynamics simulations of phakellistatin 8 in water, the decapeptide ring undergoes a conformational change from the saddle-like crystal structure to a more elongated conformation by a transition of the Tyr9 main chain from the alpha L to an extended structure. This is coupled to the loss of the NH9-O6 beta-turn hydrogen bond and the transient dissociation of the Pro7-Tyr9 side-chain packing. Furthermore, the water molecule acting as a transannular bridge forms an additional hydrogen bond with phakellistatin 8, namely with the NH group of Val5 besides those already present in the crystal structure, i.e., with the NH of Ile10 and the CO of Leu6. The alpha-turn hydrogen bond between the Phe4 amide hydrogen and the Ile10 carbonyl oxygen is always present. The solution conformations of the two cyclic decapeptides are similar, in particular in the region involving the NH4-O10 alpha turn of phakellistatin 8 and the NH5-O1 alpha turn of antamanide. The simulation results suggest that in aqueous solution the conformation of phakellistatin 8 is more extended than in the crystalline state, and on a nanosecond time scale phakellistatin 8 is more flexible than antamanide.

Antineoplastic Agents↗