The cyclic AMP receptor promoter DNA complex:A comparison of crystal and solution structure by quantitative molecular electrooptics

Abstract

The complexes formed between the cyclic AMP receptor and three different promoter DNA fragments, including a synthetic 30 bp fragment with the sequence used for determination of the crystal structure, have been analysed in solution by measurements of the electric dichroism (ED) at an ionic strength of 105 mM, using a special instrument based on cable discharge. The ED of the protein is negligible and, thus, the ED of the complexes is determined by the DNA and its orientation relative to the protein. The complex formed between the cyclic AMP receptor with the 30 bp fragment is characterized by a positive ED, indicating that the electric dipole is perpendicular relative to the direction of the helix; moreover, the dipole changes its nature from an induced one for the free DNA to a permanent one of 3.0×10−27 Cm for the complex; both the limiting value of the ED +0.3 and the dichroism decay time constant of 62 ns found for the complex (free DNA: 52 ns; 20°C) demonstrate bending of the DNA double helix. All these parameters are calculated quantitatively from the crystal structure: bead model simulations are used to derive the coefficients of rotational diffusion and to define the center of diffusion, which is the reference for calculation of the dipole vector; the dipole vector is then the basis for calculation of the limit value of the dichroism; the time constants are derived from the diffusion coefficients of the bead model The calculated parameters are in very satisfactory agreement with the experimental ones, demonstrating agreement of the structures in the crystal and in solution with respect to their essential features. These results also demonstrate the utility of electrooptical procedures for a quantitative comparison of crystal or model structures with structures in solution. While the crystal structure has been determined only for the complex with the 30 bp promoter fragment, it has been relatively simple to extend measurements of the ED to complexes formed with a 40 bp and a 203 bp promoter fragment: the data obtained for a 40 bp fragment with the consensus binding sequence are quite similar to those obtained for the 30 bp promoter, whereas the data obtained for the 203 bp promoter clearly show a much higher degree of protein induced bending with a bending angle of ∼180°.