Synthesis of a tetranitrosyl iron complex with unique structure and properties as an inhibitor of phosphodiesterases.

A novel neutral tetranitrosyl iron complex {[Fe(H2O)4]2+[FeR2(NO)2]22-}·4H2O (1) with R = 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyls (C7H5N4S), which is a supramolecular ensemble, has been synthesized and studied. As follows from X-ray diffraction analysis, this is an octahedral Fe2+complex (Lewis acid) with two monoanionic dinitrosyl groups [FeR2(NO)2]- (Lewis base) and 4 water molecules as the ligands. As follows from Mössbauer spectra, the coordinating Fe2+ ion is in a low-spin state S = 0, and the dinitrosyl Fe+ ion is in a low-spin state S = 1/2. According to the data of EPR spectroscopy, mass-spectrometry and amperometry, complex 1 in solution forms dinitrosyl particles of [Fe(C7H6N4S-H)2(NO)2]- composition, which are responsible for NO generation. In addition, complex 1 was shown to be a 5-6 times more efficient phosphodiesterase (PDE) inhibitor at 5 × 10-5 M and 10-4 M concentrations than its thioligand. Probable binding sites of the [FeR2(NO)2]- ligand for the bovine PDE1B model have been determined by molecular docking and quantum-chemical calculations.

On relation between the free-energy perturbation and Bennett's acceptance ratio methods: Tracing the influence of the energy gap.

The "double-end" free-energy perturbation (DEFEP) expression, as the Taylor expansions show, presents an asymptotic solution for Bennett's acceptance ratio (BAR) method at large energy gaps. Iterative self-consistent calculations for solving the BAR equation oscillate between two energy values in such a case, and only using the DEFEP result as a first-guess yields formal convergence of the self-consistence procedure. The DEFEP estimate also provides a good starting point for the iterative procedure of BAR for the whole range of state overlap. Microscopic force field molecular dynamics simulations of the hydration free energies for transformation O(+)-->O(-) support these data. The simulations also prove robustness of the multistage perturbation schemes as compared with single-stage calculations. The observed difference between the BAR and DEFEP results has a maximum at intermediate values of energy gaps and is getting smaller for energy gaps less than 10-15 kT.

Ions and blockers in potassium channels: insights from free energy simulations.

Potassium ion channels enable efficient and selective permeation of K+ ions across nonpolar biological membranes. Here we review the results of recent free energy calculations related to the permeation of monovalent cations through K+ channels and to the channel inhibition by blocker compounds. In particular, the progress in computational studies of the bacterial KcsA channel is discussed.

K(+)/Na(+) selectivity of the KcsA potassium channel from microscopic free energy perturbation calculations.

Microscopic molecular dynamics free energy perturbation calculations of the K(+)/Na(+) selectivity in the KcsA potassium channel, based on its experimental three-dimensional structure, are reported. The relative binding free energies for K(+) and Na(+) in the most relevant ion occupancy states of the four-site selectivity filter are calculated. The previously proposed mechanism for ion permeation through the KcsA channel is predicted, in agreement with available experimental data, to have a significant selectivity for K(+) over Na(+). The calculations also show that the individual 'binding site' selectivities are generally not additive and the doubly loaded states of the filter thus display cooperative effects. The only site that is not K(+) selective is that which is located at the entrance to the internal water cavity, suggesting the possibility that internal Na(+) could block outward currents.

Mechanisms of tetraethylammonium ion block in the KcsA potassium channel.

We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.

A computational study of ion binding and protonation states in the KcsA potassium channel.

We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.

A fast method for the quantitative estimation of the distribution of hydrophobic and hydrophilic segments in alpha-helices of membrane proteins.

The work presents a fast quantitative approach for estimating the orientations of hydrophilic and hydrophobic regions in the helical wheels of membrane-spanning alpha-helices of transmembrane proteins. The common hydropathy analysis provides an estimate of the integral hydrophobicity in a moving window which scans an amino acid sequence. The new parameter, orientation hydrophobicity, is based on the estimate of hydrophobicity of the angular segment that scans the helical wheel of a given amino acid sequence. The corresponding procedure involves the treatment of transmembrane helices as cylinders with equal surface elements for each amino acid residue. The orientation hydrophobicity, P(phi), phi = 0-360 degrees, of a helical cylinder is given as a sum of hydrophobicities of individual amino acids which are taken as the S-shaped functions of the angle between the centre of amino acid surface element and the centre of the segment. Non-zero contribution to P(phi) comes only from the amino acids belonging to the angular segment for a given angle phi. The size of the angular segment is related to the size of the channel pore. The amplitudes of amino acid S-functions are calibrated in the way that their maximum values (reached when the amino acid is completely exposed into the pore) are equal to the corresponding hydropathy index in the selected scale (here taken as Goldman-Engelman-Steitz hydropathy scale). The given procedure is applied in the studies of three ionic channels with well characterized three-dimensional structures where the channel pore is formed by a bundle of alpha-helices: cholera toxin B, nicotinic acetylcholine homopentameric alpha7 receptor, and phospholamban. The estimated maximum of hydrophilic properties at the helical wheels are in a good agreement with the spatial orientations of alpha-helices in the corresponding channel pores.

Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction.

The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu(172). Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 +/- 1. 3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.

Ion permeation mechanism of the potassium channel.

Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol(-1). Other putative permeation pathways can be excluded because they would involve states that are too high in energy.

The application of stereolithography to the fabrication of accurate molecular models.

The process of stereolithography, which automatically fabricates plastic models from designs created in certain computer-aided design programs, has been applied to the production of accurate plastic molecular models. Atomic coordinates obtained from quantum mechanical calculations and from neutron diffraction data were used to locate spheres in the I-DEAS CAD program with radii proportional to the appropriate van der Waals radii. The sterolithography apparatus was used to build the models using a photosensitive liquid resin, resulting in hard plastic models that accurately represent the computed or experimental input structures. Three examples are given to illustrate how the models can be used to interpret experimental structure-activity data for systems of biological importance or host-guest chemistry: (1) Interpretation of kinetic data for the formation of a stable blocking complex between amiloride analogs and the epithelial sodium channel, (2) interpretation of binding and neural activity data for the interaction of certain amino acids and their analogs at the L-alanine taste receptor of the channel catfish, and (3) interpretation of shape selectivity and rate acceleration in cyclodextrin catalysis using models of the neutron diffraction structure of beta-cyclodextrin and of the transition state for the cleavage of phenyl acetate by the secondary hydroxyl oxygen of beta-cyclodextrin.

[Localization of unpaired electrons in molecules of lysozyme substrate-inhibitors. I. N-acetylglucosamine]. / Issledovanie lokalizatsii nesparennykh élektronov v molekulakh substrat-ingibitorov lizotsima. I. N-atsetilgliukozamin

Electron structure of N-acetylglucoseamine molecule and its ion-radicals has been studied by quantum-chemical methods CNDO2 and JNDO, and by ESR as well. N-acetyl group is shown to be the only electron acceptor group of AGA molecule. Phototransformations of paramagnetic centres are studied at different pH values.