Biomedical subjects

Paul Tavan

Publications and source records attributed to Paul Tavan.

12 recordsLinked to original sources

PubMed2006

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model.

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.

Animals↗

PubMed2006

Structural instability of the prion protein upon M205S/R mutations revealed by molecular dynamics simulations.

The point mutations M205S and M205R have been demonstrated to severely disturb the folding and maturation process of the cellular prion protein (PrP(C)). These disturbances have been interpreted as consequences of mutation-induced structural changes in PrP, which are suggested to involve helix 1 and its attachment to helix 3, because the mutated residue M205 of helix 3 is located at the interface of these two helices. Furthermore, current models of the prion protein scrapie (PrP(Sc)), which is the pathogenic isoform of PrP(C) in prion diseases, imply that helix 1 disappears during refolding of PrP(C) into PrP(Sc). Based on molecular-dynamics simulations of wild-type and mutant PrP(C) in aqueous solution, we show here that the native PrP(C) structure becomes strongly distorted within a few nanoseconds, once the point mutations M205S and M205R have been applied. In the case of M205R, this distortion is characterized by a motion of helix 1 away from the hydrophobic core into the aqueous environment and a subsequent structural decay. Together with experimental evidence on model peptides, this decay suggests that the hydrophobic attachment of helix 1 to helix 3 at M205 is required for its correct folding into its stable native structure.

Computer Simulation↗

PubMed2005

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance.

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.

Aminobenzoates↗

PubMed2005

Molecular dynamics simulations indicate a possible role of parallel beta-helices in seeded aggregation of poly-Gln.

The molecular structures of amyloid fibers characterizing neurodegenerative diseases such as Huntington's or transmissible spongiform encephalopathies are unknown. Recently, x-ray diffraction patterns of poly-Gln fibers and electron microscopy images of two-dimensional crystals formed from building blocks of prion rods have suggested that the corresponding amyloid fibers are generated by the aggregation of parallel beta-helices. To explore this intriguing concept, we study the stability of small beta-helices in aqueous solution by molecular dynamics simulations. In particular, for the Huntington aggregation nucleus, which is thought to be formed of poly-Gln polymers, we show that three-coiled beta-helices are unstable at the suggested circular geometries and stable at a triangular shape with 18 residues per coil. Moreover, we demonstrate that individually unstable two-coiled triangular poly-Gln beta-helices become stabilized upon dimerization, suggesting that seeded aggregation of Huntington amyloids requires dimers of at least 36 Gln repeats (or monomers of approximately 54 Gln) for the formation of sufficiently stable aggregation nuclei. An analysis of our results and of sequences occurring in native beta-helices leads us to the proposal of a revised model for the PrP(Sc) aggregation nucleus.

Amino Acid Motifs↗

PubMed2005

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Animals↗

PubMed2004

Vibrational spectra from atomic fluctuations in dynamics simulations. I. Theory, limitations, and a sample application.

Hybrid molecular dynamics (MD) simulations, which combine density functional theory (DFT) descriptions of a molecule with a molecular mechanics (MM) modeling of its solvent environment, have opened the way towards accurate computations of solvation effects in the vibrational spectra of molecules. Recently, Wheeler et al. [ChemPhysChem 4, 382 (2002)] have suggested to compute these spectra from DFT/MM-MD trajectories by diagonalizing the covariance matrix of atomic fluctuations. This so-called principal mode analysis (PMA) allegedly can replace the well-established approaches, which are based on Fourier transform methods or on conventional normal mode analyses. By scrutinizing and revising the PMA approach we identify five conditions, which must be guaranteed if PMA is supposed to render exact vibrational frequencies. Besides specific choices of (a) coordinates and (b) coordinate systems, these conditions cover (c) a harmonic intramolecular potential, (d) a complete thermal equilibrium within the molecule, and (e) a molecular Hamiltonian independent of time. However, the PMA conditions [(c)-(d)] and [(c)-(e)] are generally violated in gas phase DFT-MD and liquid phase DFT/MM-MD trajectories, respectively. Based on a series of simple analytical model calculations and on the analysis of MD trajectories calculated for the formaldehyde molecule in the gas phase (DFT) and in liquid water (DFT/MM) we show that in both phases the violation of condition (d) can cause huge errors in PMA frequency computations, whereas the inevitable violations of conditions (c) and (e), the latter being generic to the liquid phase, imply systematic and sizable underestimates of the vibrational frequencies by PMA. We demonstrate that the huge errors, which are caused by an incomplete thermal equilibrium violating (d), can be avoided if one introduces mode-specific temperatures T(j) and calculates the frequencies from a "generalized virial" (GV) expression instead from PMA. Concerning ways to additionally remove the remaining errors, which GV still shares with PMA, we refer to Paper II of this work [M. Schmitz and P. Tavan, J. Chem. Phys. 121, 12247 (2004)].

Journal Article↗

PubMed2004

Vibrational spectra from atomic fluctuations in dynamics simulations. II. Solvent-induced frequency fluctuations at femtosecond time resolution.

The midinfrared (MIR) spectra of molecules in polar solvents exhibit inhomogeneously broadened bands whose spectral positions are shifted as compared to the gas phase. The shifts are caused by interactions with structured solvation shells and the broadenings by fluctuations of these interactions. The MIR spectra can be calculated from hybrid molecular dynamics (MD) simulations, which treat the solute molecule by density functional theory and the solvent by molecular mechanics by the so-called instantaneous normal mode analysis (INMA) or by Fourier transforming the time correlation function (FTTCF) of the molecular dipole moment. In Paper I of this work [M. Schmitz and P. Tavan, J. Chem. Phys. 121, 12233 (2004)] we explored an alternative method based on generalized virial (GV) frequencies noting, however, that GV systematically underestimates frequencies. As shown by us these artifacts are caused by solvent-induced fluctuations of the (i) equilibrium geometry, (ii) force constants, and (iii) normal mode directions as well as by (iv) diagonal and (v) off-diagonal anharmonicities. Here we now show, by analyzing the time scales of fluctuations and sample MD trajectories of formaldehyde in the gas phase and in water, that all these sources of computational artifacts can be made visible by a Fourier analysis of the normal coordinates. Correspondingly, the error sources (i) and (iii)-(v) can be removed by bandpass filtering, as long as the spectral signatures of the respective effects are well separated from the fundamental band. Furthermore, the artifacts arising from effect (ii) can be strongly diminished by a time-resolved version of the GV approach (TF-GV). The TF-GV method then yields for each mode j a trajectory of the vibrational frequency omega(j)(tmid R:tau) at a time resolution tau>tau(j), which is only limited by the corresponding oscillation time tau(j)=2pi/omega(j) and, thus, is in the femtosecond range. A correlation analysis of these trajectories clearly separates the librational motions from the conformational dynamics of the solvation shells and yields the inhomogeneously broadened MIR spectra, if the theory of motional narrowing is properly included. The MIR spectrum of formaldehyde in solution obtained by TF-GV agrees very well with the FTTCF result, if one applies the so-called "harmonic approximation" quantum correction factor and a temperature scaling to the FTTCF intensities. Also for INMA an excellent agreement is achieved if one disregards a slight INMA overestimate of linewidths.

Journal Article↗

PubMed2004

A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu(2+) complex.

Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

Algorithms↗

PubMed2004

Angular resolution and range of dipole-dipole correlations in water.

We investigate the dipolar correlations in liquid water at angular resolution by molecular-dynamics simulations of a large periodic simulation system containing about 40,000 molecules. Because we are particularly interested in the long-range ordering, we use a simple three-point model for these molecules. The electrostatics is treated both by Ewald summation and by minimum image truncation combined with a reaction field approach. To gain insight into the angular dependence of the simulated dipolar ordering we introduce a suitable expansion of the molecular pair distribution function into a set of two-dimensional correlation functions. We show that these functions enable detailed insights into the shell structure of the dipolar ordering around a given water molecule. For these functions we derive analytical expressions in the particular case in which liquid water is conceived as a dielectric continuum. Comparisons of these continuum models with the correlation functions derived from the simulations yield the key result that liquid water behaves like a continuum dielectric beyond distances of about 15 angstroms from a given water molecule. We argue that this should be a generic property of water independent of our modeling. By comparison of the results of the two different electrostatics treatments with the continuum description we show that the boundary artifacts occurring in both methods are isotropically distributed and are locally small in the respective boundary regions.

Journal Article↗

PubMed2004

Continuum description of ionic and dielectric shielding for molecular-dynamics simulations of proteins in solution.

We extend our continuum description of solvent dielectrics in molecular-dynamics (MD) simulations, which has provided an efficient and accurate solution of the Poisson equation, to ionic solvents as described by the linearized Poisson-Boltzmann (LPB) equation. We start with the formulation of a general theory for the electrostatics of an arbitrarily shaped molecular system, which consists of partially charged atoms and is embedded in a LPB continuum. This theory represents the reaction field induced by the continuum in terms of charge and dipole densities localized within the molecular system. Because these densities cannot be calculated analytically for systems of arbitrary shape, we introduce an atom-based discretization and a set of carefully designed approximations. This allows us to represent the densities by charges and dipoles located at the atoms. Coupled systems of linear equations determine these multipoles and can be rapidly solved by iteration during a MD simulation. The multipoles yield the reaction field forces and energies. Finally, we scrutinize the quality of our approach by comparisons with an analytical solution restricted to perfectly spherical systems and with results of a finite difference method.

Binding Sites↗

PubMed2003

Deactivation of rhodopsin in the transition from the signaling state meta II to meta III involves a thermal isomerization of the retinal chromophore C[double bond]D.

Light-induced isomerization of rhodopsin's retinal chromophore to the activating all-trans geometry initializes the formation of the active receptor state, Meta II. In the absence of peripheral regulatory proteins, the activity of Meta II is switched off spontaneously by two independent pathways: either by hydrolysis of the retinal Schiff base and dissociation of the light receptor into apoprotein opsin plus free retinal or by formation of Meta III, an inactive species with intact retinal protonated Schiff base absorbing at 470 nm. By FTIR spectroscopy on rhodopsin reconstituted with isotopically labeled chromophores in combination with quantum mechanical DFT calculations, we show that the deactivating step during formation of Meta III involves a thermal isomerization of the chromophore C[double bond]N, such that the chromophore in Meta III is all-trans-15-syn. This isomerization step is catalyzed by the protein environment and proceeds via Meta I, as suggested by its dependence on pH and on properties of the lipid/detergent environment of the protein. In the long term, Meta III decays likewise to opsin and free retinal by slow hydrolysis of the Schiff base.

Animals↗

PubMed2002

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation.

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis/trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.

Computer Simulation↗