Electrostatic Disorder-Induced Interactions in Inhomogeneous Dielectrics

We investigate the effect of quenched surface charge disorder on electrostatic interactions between two charged surfaces in the presence of dielectric inhomogeneities and added salt. We show that in the linear weak-coupling regime (i.e., by including mean-field and Gaussian-fluctuations contributions), the image-charge effects lead to a non-zero disorder-induced interaction free energy between two surfaces of equal mean charge that can be repulsive or attractive depending on the dielectric mismatch across the bounding surfaces and the exact location of the disordered charge distribution.Comment: 7 pages, 2 figure

Inclusion of ionic interactions in force field calculations of charged biomolecules – DNA structural transitions.

The potential of mean force (PMF) approach for treating polyion–diffuse ionic cloud interactions [D. M. Soumpasis (1984) Proceedings of the National Academy of Sciences USA81, 5116–5120] has been combined with the AMBER force field describing intramolecular interactions. The resultant generalized AMBER-PMF force field enables one to treat the conformational stabilities and structural transitions of charged biomolecules in aqueous electrolytes more realistically. For example, we have used it to calculate the relative stabilities of the B and Z conformations of d(C-G)6, and the B and heteronomous (H) conformations of dA12 · dT12, as a function of salt concentration. In the case of d(C-G)6, the predicted B–ZI transition occurs at 2.4M and is essentially driven by the phosphate-diffuse ionic cloud interactions alone as suggested by the results of earlier PMF calculations. The ZII conformer is less stable than the B form under all conditions. It is found that the helical parameters of the refined B and Z structures change with salt concentration. For example, the helical rise of B-DNA increases about 10% and the twist angle decreases by the same amount above 1M NaCl. In the range of 0.01–0.3M NaCl, the H form of dA12 · dT12 is found to be more stable than the B form and its stability increases with increasing salt concentration. The computed greater relative stability of the H conformation is likely due to noninclusion of the free energy contribution from the spine of hydration, a feature presumed to stabilize the B form of this sequence

Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths

Modeling DNA structures

For a molecule of biological importance, one expects a strong correlation between the three-dimensional structure and its biological function(s). Molecular simulations allow the prediction of physical properties of macromolecules. Many of these properties are closely related to the molecular structure. Model-building studies may thus supplement structurally low-resolution experimental data with detailed three-dimensional hypothetical atomic models. Such studies may give a consistent integral view of a wealth of experimental data. In most cases, such models will predict the outcome of certain experiments. Their actual results will often either confirm the model, be used for further refinement, or demand a major revision. Empirical force-fields provide a large amount of physicochemical knowledge concerning structural and other physical properties about various classes of molecules. They give reasonable bond distances and angles and prevent short van der Waals contacts. Difficulties arise for the prediction of large-scale structural elements. These are not only determined by short-range interactions, but also result from long-range electrostatic, hydration, and hydrophobic forces. In the case of the ionically driven DNA B-Z transition, the point of transition as a function of ionic strength and size can be correctly predicted (33). More must be done for a better understanding of the hydration forces (6). What type of questions will be reliably answered by a force-field? Relatively safe answers concern the local geometry of the molecules. If a conformation leads to strong distortions of bond distances or angles or to close van der Waals contacts, it can safely be rejected. Optimizing such unfavorable structures energetically may lead to structures showing how to avoid such distortions. More difficult are energetic questions: Which of two conformers is more stable, or what is the free energy of the substrate in the active site (63)? One cannot always be sure that the force-field provides the correct answers. Therefore, one should concentrate on questions that can be checked experimentally. The application of such concepts to model curved DNA (19, 60) and the DNA four-way junction (61) provides promising results. To explore the knowledge contained in the force-fields, several methods have been proposed. Taking advantage of structural symmetries may improve critically the convergence, while refining the target molecule or its building blocks. A numerically stable derivative for the torsion potential has been proposed. The optimization method of conjugated gradients (see Section II,B) is a powerful tool to find the way downhill toward a local minimum. To surmount barriers and escape local minima requires nonlocal optimization procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

A Novel model for saturation of ion conductivity in transmembrane channels

An important experimental finding in measuring ion currents through ion channels like the nicotinic acetylcholine receptor channel is the saturation of the current with increasing ionic strength. Practically all measurements are done at saturating ionic strength. Thus any theory which is supposed to describe the physical behavior of these channels, must predict this saturation, which appears to result from the finite volume of the site to be filled with ions. An Eyring rate theory that takes into account several ions entering the site is presented here. This new model possesses unexpected properties

[22] Modeling DNA structures : molecular mechanics and molecular dynamics

There are several tools to improve initial structural estimates of a molecule under study. The methods described in this section weight the structural models according to the approximate internal energy as a function of the atomic coordinates provided by a force field. The method of conjugated gradients is a powerful tool to find the way downhill toward a local minimum. Surmounting barriers and escaping local minima require nonlocal optimization procedures. Of the various possible choices the Bremermann method has been described in greatest detail. The central idea for the implementation of this method is its application to a small number of relevant degrees of freedom, allowing the remaining ones to relax to a local energetic minimum. Monte Carlo methods may be used to obtain statistical averaged quantities. Again the convergence of this method may be considerably increased if the randomly chosen directions mainly concentrate on the relevant directions. Molecular dynamics calculations not only provide averaged quantities but also give information about time-dependent processes.Model building studies may be used to supplement structurally low resolution experimental data with detailed three-dimensional hypothetical atomic models. Because of the strong relation between structure and function in biological molecules such models may give a consistent, integral view of a wealth of experimental data. In most cases such models will predict the outcome of certain experiments. The outcome of these experiments will often either confirm the model may be used for further refinement or even demand a major revision of the model. Coordinates obtained from X-ray fiber diffraction data or in special cases single-crystal data may provide the elements for DNA or RNA model building. Local and nonlocal optimization may be used to refine these structures and to evaluate their statistical significance as estimated by a chosen force field. Appreciable progress using nonlocal optimization procedures can only be expected if the dimensionality of the problem can be reduced sufficiently to the relevant degrees of freedom. Taking advantage of structural symmetries may critically improve the convergence while refining the target molecule or its building blocks. Monte Carlo and molecular dynamics methods allow one to calculate averaged quantities. In addition, molecular dynamics provides time evolutions of certain averages. During the simulation of certain physical properties of molecules a huge amount of data will be generated. They will provide many answers, but these answers may not always apply to the original question. So what type of questions will be reliably answered by a force field? Relatively safe answers concern the local geometry of the molecules. If a conformation leads to strong distortions of bond distances or angles or to close van der Waals contacts, this conformation can safely be rejected. Optimizing such unfavorable structures energetically may lead to structures showing how to avoid such distortions. More difficult are energetical questions: which of two conformers is more stable, or what is the free energy of the substrate in the active site? One cannot always be sure that the force field provides the correct answer. Therefore, one should pose only those questions which can be checked experimentally. Because of the many possible answers, the experiment may benefit by starting with a choice proposed by the simulation. The application of this procedure to curved DNA and the DNA four-way junction was successful