Catechol(amine)s as probes of lactoperoxidase catalytic site structure: spectroscopic and modeling studies.

Binding affinities to lactoperoxidase (LPO) of a homologous series of substituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid; dopamine, noradrenaline, adrenaline; L-3,4-dihydroxyphenylalanine] were studied by UV-visible spectroscopy and docking simulations. Dissociation constant (Kd) values were calculated by direct fitting of the experimental data and fall in a range of 3-95 mM. Thermodynamic parameters are comparable with those reported for the interaction of LPO with p-substituted phenols, suggesting a similar general mode of binding. Furthermore, the relative contributions to binding energy, described by the unimolecular constant Ku, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulations, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. This channel is characterized by a hydrophobic patch (six Phe residues) and by a charged contribution (two Glu and one His residues). All of the substrates, except caffeic acid, may approach the protein active site. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to address substrate orientation in relation to the side-chain terminal group.

Molecular mechanics analysis of Tet repressor TRP-43 fluorescence.

A 35% decrease in the fluorescence intensity of F75 TetR Trp-43 was observed upon binding of the tetracycline derivative 5a,6-anhydrotetracycline (AnTc) to the repressor. The fluorescence decay of Trp-43 in F75 TetR and in its complex with AnTc could be described by the sum of three exponential components, with lifetimes of about 6, 3, and 0.3 ns. The amplitudes, however, were markedly altered upon binding. The minimized energy mapping of Trp-43 chi 1 x chi 2 isomerization clearly indicated the existence of three main potential wells at positions (-160 degrees, -90 degrees) (rotamer I), (-170 degrees, 90 degrees) (rotamer II), and (-70, 150 degrees) (rotamer III). Our study of Trp-43 environment for each of the three rotamers suggests that the longest decay component may be assigned to rotamer II, the middle-lived component to rotamer I, and the subnanosecond component to rotamer III. The origin of the changes in the rotamer distribution upon AnTc binding is discussed. Anisotropy decays are also discussed within the framework of the rotamer model.

Conformational behaviour of the antineoplastic peptide dolastatin-10 and of two mutated derivatives.

The three-dimensional structure of dolastatin-10, an extremely potent cytostatic and antineoplastic peptide extracted from the mollusc Dolabella auricularia, has not yet been fully characterized in an experimental way. By means of a systematic conformational search of the natural peptide and of two mutated analogs, carried out both in vacuo and in aqueous solution, the present work allows to obtain insights into the conformational preferences of this remarkable compound. In addition, the ability to form intra- and intermolecular H-bonds as a function both of the sequence and of the conformation is discussed. The search for the best molecular conformations has been carried out using a molecular mechanics approach, based on the CVFF potential. Dolastatin-10 contains some unusual amino acids for which no experimental structural data are available. In order to check the reliability of the CVFF potential in predicting structures of such nonconventional amino acids, geometry optimizations have been carried out using the ab initio Hartree-Fock procedure. The CVFF parameterization is found to be adequate also for nonconventional amino acids.

Conformational behaviour and molecular similarity of some beta 1-adrenergic ligands.

The conformational behaviour of a series of aryloxypropanolamines was investigated by means of a new procedure which allows the sampling of the molecular torsional surface in a very efficient way. The combination of such a procedure with the standard molecular mechanics algorithms for the geometry optimization gives, as a result, the definition of a powerful computational scheme for the detailed analysis of the potential energy surface of complex molecules. The compounds studied show a remarkable tendency to form intra-molecular hydrogen bonds, which seem to play a key role in determining the lowest energy structures. The indices of molecular similarity proposed by Carbó, computed for the most stable conformers, do not account for differences between diastereoisomers, and, as a consequence, can hardly be used to attempt a structure-activity correlation.

A minimal multiconfigurational technique.

A direct minimization method previously presented by the authors is applied here to biconfigurational wave functions. A very moderate increasing in the time by iteration with respect to the one-determinant calculation and good convergence properties have been found. So qualitatively correct studies on singlet systems with strong biradical character can be performed with a cost similar to that required by Hartree-Fock calculations.