Biomedical subjects

Alessandra Villa

Publications and source records attributed to Alessandra Villa.

10 recordsLinked to original sources

PubMed2006

Conformational polymorphism of the PrP106-126 peptide in different environments: a molecular dynamics study.

Extensive molecular dynamic simulations (approximately 240 ns) have been used to investigate the conformational behavior of PrP106-126 prion peptide in four different environments (water, dimethyl sulfoxide, hexane, and trifluoroethanol) and under both neutral and acidic conditions. The conformational polymorphism of PrP106-126 in solution observed in the simulations supports the role of this fragment in the structural transition of the native to the abnormal form of prion protein in response to changes in the local environmental conditions. The peptide in solution is primarily unstructured. The simulations show an increased presence of helical structure in an apolar solvent, in agreement with the results from circular dichroism spectroscopy. In water solution, beta-sheet elements were observed between residues 108-112 and either residues 115-121 or 121-126. An alpha-beta transition was observed under neutral conditions. In DMSO, the peptide adopted an extended conformation, in agreement with nuclear magnetic resonance experiments.

Computer Simulation↗

PubMed2005

Structural basis for the enantioselectivity of an epoxide ring opening reaction catalyzed by halo alcohol dehalogenase HheC.

Halo alcohol dehalogenase HheC catalyzes the highly enantioselective dehalogenation of vicinal halo alcohols to epoxides, as well as the reverse reaction, the enantioselective and beta-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. To investigate this latter reaction, we determined X-ray structures of complexes of HheC with the favored and unfavored enantiomers of para-nitrostyrene oxide. The aromatic parts of the two enantiomers bind in a very similar way, but the epoxide ring of the unfavored (S)-enantiomer binds in a nonproductive inverted manner, with the epoxide oxygen and Cbeta atom positions interchanged with respect to those of the favored (R)-enantiomer. The calculated difference in relative Gibbs binding energy is in agreement with the observed loss of a single hydrogen bond in the S bound state with respect to the R bound state. Our results indicate that it is the nonproductive binding of the unfavored (S)-enantiomer, rather than the difference in affinity for the two enantiomers, that allows HheC to catalyze the azide-mediated ring opening of para-nitrostyrene oxide with high enantioselectivity. This work represents a rare opportunity to explain the enantioselectivity of an enzymatic reaction by comparison of crystallographic data on the binding of both the favored and unfavored enantiomers.

Alcohols↗

PubMed2005

A molecular dynamics study of the formation, stability, and oligomerization state of two designed coiled coils: possibilities and limitations.

The formation, relative stability, and possible stoichiometries of two (self-)complementary peptide sequences (B and E) designed to form either a parallel homodimeric (B + B) or an antiparallel heterodimeric (B + E) coiled coil have been investigated. Peptide B shows a characteristic coiled coil pattern in circular dichroism spectra at pH 7.4, whereas peptide E is apparently random coiled under these conditions. The peptides are complementary to each other, with peptide E forming a coiled coil when mixed with peptide B. Molecular dynamics simulations show that combinations of B + B and B + E readily form a dimeric coiled coil, whereas E + E does not fall in line with the experimental data. However, the simulations strongly suggest the preferred orientation of the helices in the homodimeric coiled coil is antiparallel, with interactions at the interface quite different to that of the idealized model. In addition, molecular dynamics simulations suggest equilibrium between dimers, trimers, and tetramers of alpha-helices for peptide B.

Binding Sites↗

PubMed2005

Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide.

We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modification of two hydrophobic core positions by Phe/[15N,13C]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept. This finding is also supported by molecular dynamics simulations. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in combination with NMR experiments was used for verification of the oligomerization equilibria of the coiled coil peptide.

Amino Acid Sequence↗

PubMed2005

Incorporating the effect of ionic strength in free energy calculations using explicit ions.

The incorporation of explicit ions to mimic the effect of ionic strength or to neutralize the overall charge on a system in free energy calculations using molecular dynamics simulations is investigated. The difference in the free energy of hydration between two triosephosphate isomerase inhibitors calculated at five different ion concentrations is used as an example. We show that the free energy difference can be highly sensitive to the presence of explicit ions even in cases where the mutation itself does not involve a change in the overall charge. The effect is most significant if the molecule carries a net charge close to the site mutated. Furthermore, it is shown that the introduction of a small number of ions can lead to very severe sampling problems suggesting that in practical calculations convergence can best be achieved by incorporating either no counterions or by simulating at high ionic strength to ensure sufficient sampling of the ion distribution.

Algorithms↗

PubMed2004

A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6.

Successive parameterizations of the GROMOS force field have been used successfully to simulate biomolecular systems over a long period of time. The continuing expansion of computational power with time makes it possible to compute ever more properties for an increasing variety of molecular systems with greater precision. This has led to recurrent parameterizations of the GROMOS force field all aimed at achieving better agreement with experimental data. Here we report the results of the latest, extensive reparameterization of the GROMOS force field. In contrast to the parameterization of other biomolecular force fields, this parameterization of the GROMOS force field is based primarily on reproducing the free enthalpies of hydration and apolar solvation for a range of compounds. This approach was chosen because the relative free enthalpy of solvation between polar and apolar environments is a key property in many biomolecular processes of interest, such as protein folding, biomolecular association, membrane formation, and transport over membranes. The newest parameter sets, 53A5 and 53A6, were optimized by first fitting to reproduce the thermodynamic properties of pure liquids of a range of small polar molecules and the solvation free enthalpies of amino acid analogs in cyclohexane (53A5). The partial charges were then adjusted to reproduce the hydration free enthalpies in water (53A6). Both parameter sets are fully documented, and the differences between these and previous parameter sets are discussed.

Algorithms↗

PubMed2003

Understanding binding affinity: a combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin.

The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para position of the benzamidinium ion. They show small differences in relative binding affinity but large compensating differences in enthalpy and entropy. Binding affinity decreases with increased branching at the first carbon but increases with increasing the length of a linear alkyl substituent, suggesting that steric hindrance and hydrophobic interactions play dominant roles in binding. Structural analysis showed that the backbone of the enzyme was unaffected by the change of the para substituent. In addition, binding does not correlate strongly with octanol/water partition data. To further characterize this system, the change in the heat capacity on binding, the change in solvent-accessible surface area on binding, the effect of inhibitor binding on the hydration of the active site, the pK(a) of His57, and interactions within the catalytic triad have been investigated. Although the changes in inhibitor structure are small, it is demonstrated that simple concepts such as steric hindrance, hydrophobicity, and buried surface area are insufficient to explain the binding data. Other factors, such as access to the binding site and the cost of dehydration of the active site, are of equal or greater importance.

Benzamidines↗

PubMed2003

Sampling and convergence in free energy calculations of protein-ligand interactions: the binding of triphenoxypyridine derivatives to factor Xa and trypsin.

The binding of a set of 10 triphenoxypyridine derivatives to two serine proteases, factor Xa and trypsin, has been used to analyze factors related to sampling and convergence in free energy calculations based on molecular dynamics simulation techniques. The inhibitors investigated were initially proposed as part of the Critical Assessment of Techniques for Free Energy Evaluation (CATFEE) project for which no experimental results nor any assessment of the predictions submitted by various groups have ever been published. The inhibitors studied represent a severe challenge for explicit free energy calculations. The mutations from one compound to another involve up to 19 atoms, the creation and annihilation of net charge and several alternate binding modes. Nevertheless, we demonstrate that it is possible to obtain highly converged results (+/- 5-10 kJ/mol) even for such complex multi-atom mutations by simulating on a nanosecond time scale. This is achieved by using soft-core potentials to facilitate the creation and deletion of atoms and by a careful choice of mutation pathway. The results show that given modest computational resources, explicit free energy calculations can be successfully applied to realistic problems in drug design.

Binding Sites↗

PubMed2002

Conformational characterization of lanthanide(III)-DOTA complexes by ab initio investigation in vacuo and in aqueous solution.

The conformational behavior of four [Ln(DOTA)(H(2)O)](-) systems (Ln = La, Gd, Ho, and Lu) has been characterized by means of ab initio calculations performed in vacuo and in aqueous solution, the latter by using the polarizable continuum model (PCM). Calculated molecular geometries and conformational energies of the [Ln(DOTA)(H(2)O)](-) systems, and interconversion mechanisms, barriers, and (13)C NMR spectra of the [Lu(DOTA)](-) complex are compared with experimental values. For each system, geometry optimizations, performed in vacuo and in solution at the HF/3-21G level and using a 46+4f(n) core electron effective core potential (ECP) for lanthanides, provide two minima corresponding to a square antiprismatic (A) and an inverted antiprismatic (IA) coordination geometry. All the systems are nonacoordinated, with the exception of the IA isomer of the Lu complex that, from in solution calculations, is octacoordinated, in agreement with experimental data. On comparing the in vacuo relative free energies calculated at different theory levels it can be seen that the nonacoordinated species dominates at the beginning of the lanthanide series while the octacoordinated one does so at the end. Furthermore, on passing along the series the IA isomer becomes less and less favored with respect to A and for the Lu complex a stabilization of the IAisomer is observed in solution (but not in vacuo), in agreement with the experimental data. Investigation of the A<-->IA isomerization process in the [Lu(DOTA)](-) system provides two different interconversion mechanisms: a single-step process, involving the simultaneous rotation of the acetate arms, and a multistep path, involving the inversion of the cyclen cycle configuration. While in vacuo the energy barrier for the acetate arm rotation is higher than that involved in the ring inversion (23.1 and 13.1 kcal mol(-)(1) at the B3LYP/6-311G level, respectively), in solution the two mechanisms present comparable barriers (14.7 and 13.5 kcal mol(-)(1)), in fairly good agreement with the experimental values. The NMR shielding constants for the two isomers of the [Lu(DOTA)](-) complex have been calculated by means of the ab initio GIAO and CSGT methods, and using a 46-core-electron ECP for Lu. The calculated (13)C NMR chemical shifts are in close agreement with the experimental values (rms 3.3 ppm, at the HF/6-311G level) and confirm the structural assignment of the two isomers based on experimental NMR spectra in solution. The results demonstrate that our computational approach is able to predict several physicochemical properties of lanthanide complexes, allowing a better characterization of this class of compounds for their application as contrast agents in medical magnetic resonance imaging (MRI).

Contrast Media↗

PubMed2002

Calculation of the free energy of solvation for neutral analogs of amino acid side chains.

The ability of the GROMOS96 force field to reproduce partition constants between water and two less polar solvents (cyclohexane and chloroform) for analogs of 18 of the 20 naturally occurring amino acids has been investigated. The estimations of the solvation free energies in water, in cyclohexane solution, and chloroform solution are based on thermodynamic integration free energy calculations using molecular dynamics simulations. The calculations show that while the force field reproduces the experimental solvation free energies of nonpolar analogs with reasonable accuracy the solvation free energies of polar analogs in water are systematically overestimated (too positive). The dependence of the calculated free energies on the atomic partial charges was also studied.

Algorithms↗