Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria

Different microscopic and semimicroscopic approaches for calculations of electrostatic energies in macromolecules are examined. This includes the Protein Dipoles Langevin Dipoles (PDLD) method, the semimicroscopic PDLD (PDLD/S) method, and a free energy perturbation (FEP) method. The incorporation of these approaches in the POLARIS and ENZYMIX modules of the MOLARIS package is described in detail. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions, as well as the effects of the ionic strength and external pH. Determination of the hydrophobic energy involves an approach that considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. Ionic strength effects are studied by modeling the ionic environment around the given system using a grid of residual charges and evaluating the relevant interaction using Coulomb's law with the dielectric constant of water. The performance of the FEP calculations is significantly enhanced by using special boundary conditions and evaluating the long-range electrostatic contributions using the Local Reaction Field (LRF) model. A diverse set of electrostatic effects are examined, including the solvation energies of charges in proteins and solutions, energetics of ion pairs in proteins and solutions, interaction between surface charges in proteins, and effect of ionic strength on such interactions, as well as electrostatic contributions to binding and catalysis in solvated proteins. Encouraging results are obtained by the microscopic and semimicroscopic approaches and the problems associated with some macroscopic models are illustrated. The PDLD and PDLD/S methods appear to be much faster than the FEP approach and still give reasonable results. In particular, the speed and simplicity of the PDLD/S method make it an effective strategy for calculations of electrostatic free energies in interactive docking studies. Nevertheless, comparing the results of the three approaches can provide a useful estimate of the accuracy of the calculated energies. © 1993 John Wiley & Sons, Inc.

Microscopic and semimicroscopic calculations of electrostatic energies in proteins by the POLARIS and ENZYMIX programs

One of the most direct benchmarks for electrostatic models of macromolecules is provided by the pKa's of ionizable groups in proteins. Obtaining accurate results for such a benchmark presents a major challenge. Microscopic models involve very large opposing contributions and suffer from convergence problems. Continuum models that consider the protein permanent dipoles as a part of the dielectric constant cannot reproduce the correct self-energy. Continuum models that treat the local environment in a semi-microscopic way do not take into account consistently the protein relaxation during the charging process. This work describes calculations of pKa's in protein in an accurate yet consistent way, using the semi-microscopic version of the protein dipoles Langevin dipoles (PDLD) model, which treats the protein relaxation in the microscopic framework of the linear response approximation. This approach allows one to take into account the protein structural reorganization during formation of charges, thus reduci...

Consistent Calculations of pKa's of Ionizable Residues in Proteins: Semi-microscopic and Microscopic Approaches

A sequence of ordered solvent peaks in the electron density map of the minor groove region of ApT-rich tracts of the double helix is a characteristic of B-form DNA well established from crystallography. This feature, termed the “spine of hydration”, has been discussed as a stabilizing feature of B-DNA, the structure of which is known to be sensitive to environmental effects. Nanosecond-range molecular dynamics simulations on the DNA duplex of sequence d(CGCGAATTCGCG) have been carried out, including explicit consideration of ∼4000 water molecules and 22 Na+ counterions, and based on the new AMBER 4.1 force field with the particle mesh Ewald summation used in the treatment of long-range interactions. The calculations support a dynamical model of B-DNA closer to the B form than any previously reported. Analysis of the dynamical structure of the solvent revealed that, in over half of the trajectory, a Na+ ion is found in the minor groove localized at the ApT step. This position, termed herein the “ApT pocket...

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Intrusion of Counterions into the Spine of Hydration in the Minor Groove of B-DNA: Fractional Occupancy of Electronegative Pockets

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.

Activation and detoxication of aflatoxin B1

The hydrogen ion concentration in the vicinity of DNA was mapped out within the Poisson-Boltzmann approximation. Experimental conditions were modeled by assuming Na-DNA to be solvated in a buffer solution containing 45 mM Tris and 3 mM Mg cations at pH 7.5. Three regions of high H+ concentration (greater than 10 microM) are predicted: one throughout the minor groove of DNA and two localized in the major groove near N7 of guanine and C5 of cytosine for a G.C base pair. These acidic domains correlate well with the observed covalent binding sites of benzo[a]pyrene epoxide (N2 of guanine) and of aflatoxin B1 epoxide (N7 of guanine), chemical carcinogens that presumably undergo acid catalysis to form highly reactive carbocations that ultimately bind to DNA. It is suggested that these regions of high H+ concentration may also be of concern in understanding interactions involving proteins and noncarcinogenic molecules with or near nucleic acids.

Acidic domains around nucleic acids.

The counterion density and the condensation region around DNA have been examined as functions of both ion size and added-salt concentration using Metropolis Monte Carlo (MC) and Poisson–Boltzmann (PB) methods. Two different definitions of the “bound” and “free” components of the electrolyte ion atmosphere were used to compare these approaches. First, calculation of the ion density in different spatial regions around the polyelectrolyte molecule indicates, in agreement with previous work, that the PB equation does not predict an invariance of the surface concentration of counterions as electrolyte is added to the system. Further, the PB equation underestimates the counterion concentration at the DNA surface, compared to the MC results, the difference being greatest in the grooves, where ionic concentrations are highest. If counterions within a fixed radius of the helical axis are considered to be bound, then the fraction of polyelectrolyte charge neutralized by counterions would be predicted to increase as the bulk electrolyte concentration increases.



A second categorization—one in which monovalent cations in regions where the average electrostatic potential is ledd than −kT are considered to be bound—provides an informative basis for comparison of MC and PB with each other and with counterion-condensation theory. By this criterion, PB calculations on the B from of DNA indicate that the amount of bound counterion charge per phosphate group is about .67 and is independent of salt concentration. A particularly provocative observatiob is that when this binding criterion is used, MC calculations quantitatively reproduce the bound fraction predicated by counterion-condensation theory for all-atom models of B-DNA and A-DNA as well as for charged cylindera of varying lineat charge densities. For example, for B-DNA and A-DNA, the fractions of phosphate groups neutralized by 2 A hard sphere counterions are 0.768 and .817, respectively. For theoretical studies, the rediys enclosing the region in which the electrostatic potential is calculated studies, the radius enclosing the region in which the electrostatic potential is calculated to be less than −kT is advocated s a more suitable binding or condensation radius that enclosing the fraction of counterions given by (1 – ξ−1). A comparsion of radii calculated using both of these definitions is presented. © 1994 John Wiley & Sons, Inc.

Monte Carlo and Poisson-Boltzmann calculations of the fraction of counterions bound to DNA.

A technique for modeling the structured environmental charge distribution about isolated polyions of arbitrary geometry is presented and applied to B-DNA. It describes the three-dimensional variation of the continuous space charge and allows estimation of local electrostatic potentials and fields that the electrolytic environment induces at nuclei of the polyion. Calculations involve an iterative solution to the set of equations coupling electrostatic potential and average charge density in space. By dividing the region around a DNA segment into finite volume elements, sets of numerically stable atmospheric charge densities have been obtained over a range of concentrations of added monovalent salt. Results are in good agreement with those of Poisson-Boltzmann calculations on comparable systems and are consistent with findings from Monte Carlo simulations of DNA.

Calculations of the spatial distribution of charge density in the environment of DNA.

A solution to the three-dimensional Poisson-Boltzmann equation, generalized to include the finite size of the ions, is presented for the environment of DNA in the B- and Z-conformations. The results clearly indicate that despite the lower linear charge density of the left-handed Z-conformer, there is a higher concentration of Na+ at the immediate surface of the Z-from than at the surface of the B-form. The average concentration of counterions within a 12-A radius of the DNA is, nonetheless, higher for the B- than for the Z-form. Calculations of the electrostatic interactions of these conformers with an environment of 0.01M monovalent salt show that the salt exerts a greater stabilizing effect on the left-handed conformer than on the right-handed form.

George R. Pack

Papers

Acidic domains around nucleic acids.

Monte Carlo and Poisson-Boltzmann calculations of the fraction of counterions bound to DNA.

Calculations of the spatial distribution of charge density in the environment of DNA.

Generalized Poisson-Boltzmann calculation of the distribution of electrolyte ions around the B- and Z-conformers of DNA.

DNA-Mediated Acid Catalysis: Calculations of the Rates of DNA-Catalyzed Hydrolyses of Diol Epoxides1