Kinetics of the B-A transition of DNA: analysis of potential contributions to a reaction barrier.

Because of open problems in the relation between results obtained by relaxation experiments and molecular dynamics simulations on the B-A transition of DNA, relaxation measurements of the B-A dynamics have been extended to a wider range of conditions. Field-induced reaction effects are measured selectively by the magic angle technique using a novel cell construction preventing perturbations from cell window anisotropy. The kinetics was recorded for the case of poly[d(AT)] up to the salt concentration limit of 4.

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 84-859 bp has been analyzed in aqueous solutions upon reduction of the water activity. Rotational diffusion times τ (d) measured by electric dichroism at different water activities with a wide variation of viscosities are normalized to values τ (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 ≤1 mM.

Recognition of operator DNA by Tet repressor.

The recognition of operator DNA by Tet repressor was analyzed by fluorescence stopped flow measurements. The main part of the fluorescence change observed for the reaction of the repressor with operator DNA reflects a second-order binding reaction including the expected concentration dependence. Global fitting of transients measured at different concentrations reveal at least one intramolecular step in addition to the bimolecular step.

Electro-optical analysis of macromolecular structure and dynamics.

Electro-optical effects are induced by external electric field pulses applied to solutions or suspensions and are recorded by various optical techniques. These effects are very useful for the characterization of macromolecular structures and their dynamics in solution. One of the field-induced effects is alignment of molecular dipoles, which can be detected at a very high sensitivity by measurements of the dichroism or the birefringence.

Allosteric control of cAMP receptor binding dynamics.

The intrinsic fluorescence of the cyclic AMP receptor is a sensitive indicator of the reaction with DNA, but signals are perturbed by a photoreaction. A ratio procedure is shown to be useful for correction. The reaction of the protein with DNA indicated by corrected transients extends over a broad time range not only at low salt concentrations but also at physiological salt concentrations.

Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific sites.

The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by an increase of both rise- and decay-time constants.

Electric birefringence at small angles from crossed position: enhanced sensitivity and special effects.

Measurements of electric birefringence with increased sensitivity are possible using lasers with high intensity and stability, provided that perturbations resulting from stray light and strain in cell windows can be reduced. A new type of cell window is designed for minimal strain and is used in a standard birefringence setup with optimized components. The new instrument is characterized by a stray-light constant of 2 × 10(-7) and a negligible residual birefringence.

Allosteric control of promoter DNA bending by cyclic AMP receptor and cyclic AMP.

The structure of the cyclic AMP receptor-promoter complex in solution was studied in the range of 0.2-50 microM cAMP by measurements of the electric birefringence at 0.1 M salt using a lac promoter DNA with 121 bp and with the CAP binding site at its center.

Effects of hydrodynamic coupling on electro-optical transients.

The effects resulting from coupling between translational and rotational diffusion on electro-optical transients were analyzed by Brownian dynamics simulations. Diffusion tensors, including translational-rotational coupling tensors, were derived from bead model simulations. Optical and electrical parameters were assigned according to models and experimental data.

Unique physical signature of DNA curvature and its implications for structure and dynamics.

A particularly sensitive birefringence technique is used to analyze a curved DNA fragment with 118 bp and a standard DNA with 119 bp. At salt concentrations from 0.5 to 10 mM, both fragments show the usual negative stationary birefringence and monotonic transients - differences are relatively small.

The nature of "unusual" electro-optical transients observed for DNA.

Unusual electro-optical transients have been observed for many different polymers and colloidal systems. These effects provoked serious confusion, because a simple-minded interpretation can be completely misleading. The case of double helical DNA is of particular interest, because DNA has been studied in more detail than other systems and because of its biological function.

Strong effect of hydrodynamic coupling on the electric dichroism of bent rods.

The effect of hydrodynamic coupling on the spatial orientation of rigid bent rods in electric fields has been analyzed by Brownian dynamics simulations. Bead models for smoothly bent rods were constructed with dimensions of DNA double helices, and established simulation procedures were used to calculate their diffusion tensor, including the translational-rotational coupling tensor. The electric and optical parameters were assigned on the basis of known properties of double helices.

The dynamics of the B-A transition of natural DNA double helices.

The dynamics of the B-A transition of DNA double helices with different GC contents and various chain lengths has been characterized by an electric field pulse technique. The field-induced B-A reaction is separated from orientation effects using the magic angle technique. Amplitudes reflecting the B-A reaction are observed selectively in the limited range of ethanol contents, where CD spectra demonstrate the B-A transition.

Dynamics of the B-A transition of DNA double helices.

Although the transition from the B-DNA double helix to the A-form is essential for biological function, as shown by the existence of the A-form in many protein-DNA complexes, the dynamics of this transition has not been resolved yet. According to molecular dynamics simulations the transition is expected in the time range of a few nanoseconds. The B-A transition induced by mixing of DNA samples with ethanol in stopped flow experiments is complete within the deadtime, showing that the reaction is faster than approximately 0.

Strong bending of purple membranes in the M-state.

Structure changes of purple membranes during the photocycle were analysed in solution by measurements of the electric dichroism. The D96N-mutant was used to characterize the M-state at neutral pH. The transition from the resting state to 61% photo-stationary M-state is associated with a strong reduction of the dichroism decay time constant by a factor of approximately 2.