Molecular dynamics simulations of the free and inhibitor-bound cruzain systems in aqueous solvent: insights on the inhibition mechanism in acidic pH.

The major cysteine protease of Trypanosoma cruzi, cruzain (CRZ), has been described as a therapeutic target for Chagas' disease, which affects millions of people worldwide. Thus, a series of CRZ inhibitors has been studied, including a new competitive inhibitor, Nequimed176 (NEQ176). Nevertheless, the structural and dynamic basis for CRZ inhibition remains unclear. Hoping to contribute to this ever-growing understanding of timescale dynamics in the CRZ inhibition mechanism, we have performed the first study using 100 ns of molecular dynamics (MD) simulations of two CRZ systems in an aqueous solvent under pH 5.5: CRZ in the apo form (ligand free) and CRZ complexed to NEQ176. According to the MD simulations, the enzyme adopts an open conformation in the apo form and a closed conformation in the NEQ176-CRZ complex. We also suggest that this closed conformation is related to the hydrogen-bonding interactions between NEQ176 and CRZ, which occurs through key residues, mainly Gly66, Met68, Asn69, and Leu160. In addition, the cross-correlation analysis shows evidence of the correlated motions among Ala110-Asp140, Leu160-Gly189, and Glu190-Gly215 subdomains, as well as, the movements related to Ala1-Thr59 and Asp60-Pro90 regions seem to be crucial for CRZ activity.

Self-similarity and protein compactness.

The hydrophobic effect is the major factor that drives a protein toward collapse and folding. As a consequence of the folding process a hydrophobic core is shielded by the solvent-accessible surface area of the protein. We analyze the solvent-accessible surface area of 1825 nonhomolog protein chains deposited in the Brookhaven Protein Data Bank. This solvent-accessible surface area presents an intrinsic self-similarity behavior. The comparison between the accessible surface area as function of the number of amino acids and the accessible surface area as function of gyration radius supplies a measure of the scaling exponent close to the one observed by volume as function of radius of gyration or by mass-size exponent. The present finding indicates that the fractal analysis describes the protein compactness as an object packing between random spheres in percolation threshold and crumpled wires.

New stochastic strategy to analyze helix folding.

We propose an alternative stochastic strategy to search secondary structures based on the generalized simulated annealing (GSA) algorithm, by using conformational preferences based on the Ramachandran map. We optimize the search for polypeptide conformational space and apply to peptides considered to be good alpha-helix promoters above a critical number of residues. Our strategy to obtain conformational energies consist in coupling a classical force field (THOR package) with the GSA procedure, biasing the Phi x Psi backbone angles to the allowed regions in the Ramachandran map. For polyalanines we obtained stable alpha-helix structures when the number of residues were equal or exceeded 13 amino acids residues. We also observed that the energy gap between the global minimum and the first local minimum tends to increase with the polypeptide size. These conformations were generated by performing 2880 stochastic molecular optimizations with a continuum medium approach. When compared with molecular dynamics or Monte Carlo methods, GSA can be considered the fastest.

Multifractality, Levinthal paradox, and energy hypersurface.

Multifractal properties in the potential energy hypersurface of polypeptides and proteins are investigated. Characteristic multifractal behavior for different molecular systems is obtained from the f(alpha) spectra. The analysis shows that the dimension of the phase space of the problem influences the accessibility to different parts of the potential energy hypersurface. Also, we show that it is necessary to take into account the H-bond formation between amino acids in the conformational-folding search. The present findings indicate that the f(alpha) function describes some structural properties of a protein. The behavior of the f(alpha) spectra gives an alternative explanation about the Levinthal paradox. Furthermore, the anomalous temperature dependence of the Raman spin-lattice relaxation rates can be related to the perturbations in the secondary structures.

Molecular dynamics simulation of alpha-melanocyte stimulating hormone in a water-membrane model interface.

The conformation of the tridecapeptide alpha-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant epsilon, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation.

Molecular dynamics at a cytoplasm/membrane interface of a signal sequence from an E. coli maltoporin.

We used a recently developed software that mimics a cytoplasm/membrane environment, with an interface separating two continuous media of different dielectric constants (1). This software has been designed to allow modelling of different kinds of molecules of biological interest such as proteins and drugs, in each of the isolated continuous media, as well as in their interactions with membrane-like structures, making use of the dielectric discontinuity represented by the interface. In the present study we have applied this program ("THOR") to model a polypeptide sequence corresponding to a 50% active mutant of the signal sequence of the lamB gene product of E. coli, known as maltoporin or lambda receptor (2). The peptide was first submitted to optimization of its molecular geometry followed by molecular dynamics in water (epsilon = 80) until thermalization was achieved. The conformation evolved from a rather extended random conformation to increasingly folded structures. The presence of the dielectric discontinuity induced the movement of the molecule's center of mass from water towards the interface. The entry of the peptide into the lower dielectric constant medium (epsilon = 2) through the interface was paralleled by a decrease in the total potential energy, indicating the affinity of the peptide for the lipid-mimetic phase.

EPR study of melanin-protein interaction: photoinduced free radicals and progressive microwave power saturation.

The photo-induction of free radicals in synthetic L-dihydroxyphenylalanine (L-DOPA) melanin in the presence of bovine serum albumin (BSA) was studied by electron paramagnetic resonance (EPR) spectroscopy. By monitoring the signal intensities and progressive microwave power saturation it was shown that L-DOPA melanin in solution behaves as a single macromolecule, interacting with BSA and molecular oxygen. In the absence of oxygen, the EPR signal of L-DOPA melanin was homogeneously broadened; the magnetic interaction with oxygen induced inhomogeneous broadening. In aqueous solution, the presence of BSA decreased the accessibility of oxygen to paramagnetic centres in the melanin. On UV-visible illumination, the presence of BSA modified the rates of formation and decay of photoinduced free radicals, resulting in a net enhancement of the EPR signal compared with that observed in pure L-DOPA melanin.