The investigation of the effects of counterions in protein dynamics simulations.

Molecular simulations able to exactly represent solvated charged proteins are helpful in understanding protein dynamics, structure and function. In the present study we have used two different starting structures of papain (a typical, stable, globular protein of intermediate net charge) and different modeling procedures to evaluate some effects of counterions in simulations. A number of configurations have been generated and relaxed for each system by various combinations of constrained simulated annealing and molecular dynamics procedures, using the AMBER force field. The analysis of trajectories shows that the simulations of solvated proteins are moderately sensitive to the presence of counterions. However, this sensitivity is highly dependent on the starting model and different procedures of equilibration used. The neutralized systems tend to evince smaller root mean square deviations regardless of the system investigated and the simulation procedure used. The results of parameterized fitting of the simulated structures to the crystallographic data, giving quantitative measure of the total charge influence on the stability of various elements of the secondary structure, revealed a clear scatter of different reactions of various systems' secondary structures to counterions addition: some systems apparently were stabilized when neutralized, while the others were not. Thus, one cannot unequivocally state, despite consideration of specific simulation conditions, whether protein secondary structures are more stable when they have neutralized charges. This suggests that caution should be taken when claiming the stabilizing effect of counterions in simulations other than those involving small, unstable polypeptides or highly charged proteins.     

Condensation of Co++ ions on chondroitin sulfate from transport coefficients and proton NMR measurements in aqueous solutions

The “condensed” counterions which characterize high-charge-density polyelectrolyte solutions can be analyzed into two subpopulations: (1) site-bound counterions and (2) atmospherically entrapped counterions. The distinction is achieved experimentally by combining the data from self-diffusion coefficient or electrical mobility measurements, which give the amount of “condensed” ions, and those from nmr, chemical shift measurements, which indicate the amount of site-bound ions. In the case of a solution of chondroitin sulfate with excess Co⁺⁺ counterions, it can be estimated that 20% of the structural charge of the polyion is neutralized by site-bound, dehydrated, condensed counterions, while a further 30% is neutralized by atmospherically entrapped, hydrated counterions.     

Electrostatics of membrane adhesion.

We consider electrical double layer interaction under the conditions typically encountered during membrane fusion. Within the physiological concentration range of monovalent electrolytes the interaction is repulsive and the Poisson-Boltzmann calculation may be used to evaluate the force. When divalent counterions are added, strong ion-ion correlations make the Poisson-Boltzmann approximation inapplicable. We use the anisotropic hypernetted chain method to show that in the presence of small amounts of divalent counterions in adsorption equilibrium with the surfaces, the double layer interaction turns into attraction. This attractive electrostatic force may be the balancing contribution controlling membrane adhesion.     

Dynamics of the DNA duplex formation studied by single molecule force measurements

DNA is partly denatured in vitro by applying a force that mechanically separates the two strands of the double helix. Sudden reduction of the imposed displacement triggers spontaneous reannealing of the molecule. The corresponding force signals are measured by optical trapping interferometry for backward steps of various amplitudes and base sequence intervals. The measured signals frequently show plateaus of varying duration at discrete values that are dependent on the base sequence. Additional measurements are performed with proteins bound to the double helix. When the opening fork encounters such a protein during mechanical unzipping, force increases until the protein is ejected. This ejection induces fast release of tension and fast unzipping. Comparing our different measurements, we find that both DNA unzipping and the relaxation of tension in DNA are faster than the formation of the double helix.     

DNA conformation and thermostability of aqueous solutions of beta-alanine and gamma-aminobutyric acid

It has been shown that at low concentrations of rare amino acids (from 10(-3) M to 10(-1) M of the substance) stechiometric complexes amino acid -- DNA are formed, which bring about partial substitution of counterions screening phosphate groups and to a change of spatial structure of DNA water molecules. The DNA-solvent molecular interactions are changed, accompanied by an abrupt decrease of helix-coil enthalpy transition which leads to the unwinding of DNA double helix. In the region of amino acid high concentrations (greater than 1-1,5 M) a rise of thermostability and winding of DNA double helix is observed. It has been established that B----C-like conformational transition stimulated by the rise of DNA thermostability is a result of counterions dehydration and the increase of effective ionic strength of the solution which is due to the rise of amino acid-zwitterions content in it.     

Hydration structure and dynamics of B- and Z-DNA in the presence of counterions via molecular dynamics simulations

Following our previous attempts at understanding the structural and dynamical properties of water and counterions hydrating nucleic acids, we have performed molecular dynamics simulations for B- and Z-DNA. In these simulations, the nucleic acids were held rigid. In the case of B-DNA, one turn of B-DNA double helix was considered in the presence of 1500 water molecules and 20 counterions (K⁺). The simulations were performed for 4.0 ps after equilibrating the system. For Z-DNA, we considered one turn of the double helix in the presence of 1851 water molecules and 24 counterions (K⁺). The simulations were carried out for 3.5 ps after equilibration. The average temperature of these simulations was ～ 360 K for Z-DNA and ～ 345 K for B-DNA. In these simulations the hydrogen atoms were explicitly taken into account. For both simulations, a fifth-order predictor-corrector was used for solving the translational equations of motion. The rotational motion of the water molecules was represented in terms of quaternion algebra and the rotational equations of motion were solved with a second-order quaternion method using a sixth-order predictor-corrector method. A time step of 0.5 · 10^?15 s was used in these simulations. The structural and the dynamical properties of water solvating the counterions, and the phosphate groups of the DNA, were computed to understand the hydration structure. Diffusion coefficients and velocity correlation functions were calculated for both ions and the water molecules. The velocity correlation functions for the ions exhibit a caged behavior. The dipole correlation functions for the water molecules indicate that the water molecules close to the helix retain the memory of their initial orientations for longer periods of time than those away from the helix. During the time period of our simulation (3–4 ps) the ion probability distributions show a well-defined pattern and suggest limited mobility for the ions, being close to the helix.     

Electrostatic free energy landscapes for DNA helix bending

Nucleic acids are highly charged polyanionic molecules; thus, the ionic conditions are crucial for nucleic acid structural changes such as bending. We use the tightly bound ion theory, which explicitly accounts for the correlation and ensemble effects for counterions, to calculate the electrostatic free energy landscapes for DNA helix bending. The electrostatic free energy landscapes show that DNA bending energy is strongly dependent on ion concentration, valency, and size. In a Na⁺ solution, DNA bending is electrostatically unfavorable because of the strong charge repulsion on backbone. With the increase of the Na⁺ concentration, the electrostatic bending repulsion is reduced and thus the bending becomes less unfavorable. In contrast, in an Mg²⁺ solution, ion correlation induces a possible attractive force between the different parts of the helical strands, resulting in bending. The electrostatically most favorable and unfavorable bending directions are toward the major and minor grooves, respectively. Decreasing the size of the divalent ions enhances the electrostatic bending attraction, causing an increased bending angle, and shifts the most favorable bending to the direction toward the minor groove. The microscopic analysis on ion-binding distribution reveals that the divalent ion-induced helix bending attraction may come from the correlated distribution of the ions across the grooves in the bending direction.     

DNA condensation by multivalent cations

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation : condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix. © 1998 John Wiley & Sons, Inc. Biopoly 44: 269–282, 1997     

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

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 Å 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.     

Dielectric behavior of polyelectrolytes. III. The role of counterion interactions

The role of the Coulomb forces between the counterions on the surface of polyelectrolytes on the dielectric response is analyzed. An estimate of the maximum dielectric increment (as a function of the number of counterions) is found as a function of the molecular length. The minimum-energy configuration of the counterions on a cylinder is found to be a double helix, suggesting the fundamental importance of electrostatic interactions in determining structure. Solutions of the dynamical equations for a few counterions indicate that a single mode dominates the relaxation which is enhanced by the inter-ion repulsions. A lower bound is found for this mode based on analysis of the system response for short lengths. Sum rules for the rates and amplitudes of the dipolar correlation function are derived and lead to an upper bound for the rate of the dominant mode. These bounds approach one another for the parameters characteristic of restriction fragments of DNA. This permits a prediction of the magnitude and time scale of the dielectric response.     

Apolipoprotein A-I helix 6 negatively charged residues attenuate lecithin-cholesterol acyltransferase (LCAT) reactivity

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.     

Growth of linear charged micelles

The electrostatics of micellar growth is reviewed and extended for solutions containing excess salt. In dilute solution the expansion of a linear micelle with increasing salt concentration is explained for a wide range of ionic strength. When the micellar charge density is very high, counterions condense nonuniformly onto the micellar rod. In that case the micelle may contract upon the addition of salt. In semidilute solutions the excluded-volume effect is an additional factor complicating the ionic strength dependence of micellar growth.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Hydration of counterions interacting with DNA double helix: a molecular dynamics study

In the present work, molecular dynamics simulations have been carried out to study the dependence of counterion distribution around the DNA double helix on the character of ion hydration. The simulated systems consisted of DNA fragment d(CGCGAATTCGCG) in water solution with the counterions Na⁺, K⁺, Cs⁺ or Mg²⁺. The characteristic binding sites of the counterions with DNA and the changes in their hydration shell have been determined. The results show that due to the interaction with DNA at least two hydration shells of the counterions undergo changes. The first hydration shell of Na⁺, K⁺, Cs⁺, and Mg²⁺ counterions in the bulk consists of six, seven, ten, and six water molecules, respectively, while the second one has several times higher values. The Mg²⁺ and Na⁺ counterions, constraining water molecules of the first hydration shell, mostly form with DNA water-mediated contacts. In this case the coordination numbers of the first hydration shell do not change, while the coordination numbers of the second one decrease about twofold. The Cs⁺ and K⁺ counterions that do not constrain surrounding water molecules may be easily dehydrated, and when interacting with DNA their first hydration shell may be decreased by three and five water molecules, respectively. Due to the dehydration effect, these counterions can squeeze through the hydration shell of DNA to the bottom of the double helix grooves. The character of ion hydration establishes the correlation between the coordination numbers of the first and the second hydration shells.

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Graphical Abstract Hydration of counterions interacting with DNA double helix

     

Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.     

Interplay between structure and dynamics in chitosan films investigated with solid-state NMR, dynamic mechanical analysis, and X-ray diffraction

Modern solid-state NMR techniques, combined with X-ray diffraction, revealed the molecular origin of the difference in mechanical properties of self-associated chitosan films. Films cast from acidic aqueous solutions were compared before and after neutralization, and the role of the counterion (acetate vs Cl(-)) was investigated. There is a competition between local structure and long-range order. Hydrogen bonding gives good mechanical strength to neutralized films, which lack long-range organization. The long-range structure is better defined in films cast from acidic solutions in which strong electrostatic interactions cause rotational distortion around the chitosan chains. Plasticization by acetate counterions enhances long-range molecular organization and film flexibility. In contrast, Cl(-) counterions act as a defect and impair the long-range organization by immobilizing hydration water. Molecular motion and proton exchange are restricted, resulting in brittle films despite the high moisture content.     

Salt dependence of the elasticity and overstretching transition of single DNA molecules

As double-stranded DNA is stretched to its B-form contour length, models of polymer elasticity can describe the dramatic increase in measured force. When the molecule is stretched beyond this contour length, it shows a highly cooperative overstretching transition. We have measured the elasticity and overstretching transition as a function of monovalent salt concentration by stretching single DNA molecules in an optical tweezers apparatus. As the sodium ion concentration was decreased from 1000 to 2.57 mM, the persistence length of DNA increased from 46 to 59 nm, while the elastic stretch modulus remained approximately constant. These results are consistent with the model of Podgornik, et al. (2000, J. Chem. Phys. 113:9343-9350) using an effective DNA length per charge of 0.67 nm. As the monovalent salt concentration was decreased over the same range, the overstretching transition force decreased from 68 to 52 pN. This reduction in force is attributed to a decrease in the stability of the DNA double helix with decreasing salt concentration. Although, as was shown previously, the hydrogen bonds holding DNA strands in a helical conformation break as DNA is overstretched, these data indicate that both DNA strands remain close together during the transition.     

Origin of DNA helical structure and its sequence dependence

Conformational analysis of DNA shows that the origin of the B-form double helix can be attributed in large part to the atomic charge pattern in the base pairs. The charge patterns favor specific helical stacking of the base pairs. Base pairs alone--without backbones--have a strong tendency to form helix, indicating that the backbones play a rather passive role in determining the basic helical structure of DNA. It is mainly the electrostatic interactions determined by the charge pattern on base pairs that stabilize a particular helical conformation. The charge pattern in the base pairs appears to be responsible for much of the sequence dependence of DNA conformation, rather than steric clashes.     

Specific helix stabilization of poly(L-glutamic acid) in mixed counterion systems

The coil–helix transitions of poly (L -glutamic acid) in aqueous alcohol solutions have been investigated for mixed counterion systems. It has been found that coexistence of two kinds of counterion species, i.e., two alkali metal counterions, alkali and alkaline earth metal, and two alkaline earth metals, specifically stabilizes or destabilizes the helix conformation depending upon the combination of the counterion species. The most striking enhancement of the helix content was observed for the combination of Li⁺ and K⁺ counterions. It has been suggested that the helix stabilization is attributed to the reduction of the free energy in the contact ion pair formation between the polymer charges and the counterions in the mixed counterion systems. © 1993 John Wiley & Sons, Inc.