Boundary conditions for free A-DNA in solution and the relation of local to global DNA structures at reduced water activity.

Because of repeated claims that A-DNA cannot exist without aggregation or condensation, the state of DNA restriction fragments with 84-859bp has been analyzed in aqueous solutions upon reduction of the water activity. Rotational diffusion times tau (d) measured by electric dichroism at different water activities with a wide variation of viscosities are normalized to values tau (c) at the viscosity of water, which indicate DNA structures at a high sensitivity. For short helices (chain lengths [Formula: see text]≤persistence length p), cooperative formation of A-DNA is reflected by the expected reduction of the hydrodynamic length; the transition to the A-form is without aggregation or condensation upon addition of ethanol at monovalent salt ≤1mM. The aggregation boundary, indicated by a strong increase of tau (c), is shifted to higher monovalent salt (≥4mM) when ethanol is replaced by trifluoroethanol. The BA transition is not indicated anymore by a cooperative change of tau (c) for [Formula: see text]p; tau (c) values for these long chains decrease upon reduction of the water activity continuously over the full range, including the BA transition interval. This suggests a non-cooperative BC transition, which induces DNA curvature. The resulting wide distribution of global structures hides changes of local length during the BA transition. Free A-DNA without aggregation/condensation is found at low-salt concentrations where aggregation is inhibited and/or very slow. In an intermediate range of solvent conditions, where the A-form starts to aggregate, a time window remains that can be used for analysis of free A-DNA in a quasi-equilibrium state

Effect of aminoacylation on tRNA conformation.

Translational diffusion coefficients have been simulated for various conformations of tRNAPhe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes

Dipole reversal in bacteriorhodopsin and separation of dipole components

The electrostatics of purple membranes has been analyzed by measurements of the electric dichroism in dc and ac fields in a broad pH range. The dc data are mainly used to characterize the permanent dipole, whereas the ac data serve as control for changes of global structure and of the induced dipole. At pH values from 8 to 3.5, the dc dichroism is negative at low field strengths and turns to positive values at higher field strengths, in qualitative agreement with the orientation function for disks having a permanent dipole moment perpendicular to the plane and an induced dipole moment in the plane. The minimum value of the dichroism, ξmin/dc , indicates the permanent dipole and shows a complex dependence on the pH but does not approach zero at any pH between 3.5 and 8, which would be expected for a simple reversal of the permanent dipole. However, the dependence of ξmin/dc on pH shows a λ point at pH 4.9, which reflects reversal of the dipole. Fitting of the stationary dichroism for E smaller or equal to 12 kV m-1 to the orientation function shows a decrease of the permanent dipole λ in the pH range 5 to about 50% of the value found at pH 7. In the same pH range, the limit dichroism, ξdc/infinity, derived from dc data also decreases to about 50% of the value at pH 7, whereas parallel measurements of the ac dichroism show almost constant ξac/infinity values. The combined observations indicate reversal of the permanent dipole, changes of disk bending, and the existence of a fluctuating dipole moment, probably resulting from bending fluctuations. Reversal of the dipole moment at pH 4.9 is confirmed by combined pH and field jump experiments. The direction of the dipole is reverted in the same pH range as the direction of stationary pH changes upon illumination, indicating an important function of the dipole for vectorial proton transfer. Comparison of experimental data with simple calculations of the protein dipole from the crystal structure indicates the existence of a large dipole component, which is directed opposite to the protein dipole at pH 7 and is probably due to a nonsymmetric distribution of charges on lipid residues. The results indicate a high degree of compensation in the electric asymmetry, which seems to be necessary for stability of purple membranes

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA.

Dichroism decay curves of DNA fragments with chain lengths in the range of 179–256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model

DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns