SPIN-LABELED NUCLEOTIDE MOBILITY IN THE BOUNDARY OF THE ECORI ENDONUCLEASE BINDING-SITE

A complex consisting of the EcoRI endonuclease site-specifically bound to spin labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein bindi ng. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking reg ion on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix cau sed by protein binding. The spectral line shapes indicate that the spi n label closest to the EcoRI endonuclease binding site, i.e., in posit ion 6, is most influenced by the binding event. The EPR data are analy zed according to a model that distinguishes between spectral effects d ue to a change in the hydrodynamic shape of the complex and those resu lting from local variations in the spin-label mobility as characterize d by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 +/- 0.01 for all three oligomers as w ell as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S=0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 lab eled material, molecular dynamics (MD) simulations were used to explor e the space accessible to the probes in positions 6, 9, and II. MD res ults gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the D NA at this location possibly corresponding to a bend induced by protei n binding. The observation that the spectral changes are small indicat es the absence of any significant structural disruption being propagat ed along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodyn amic theory in view of the significant increase in molecular volume up on protein binding suggests that there are additional relaxation proce sses involving the protein and nucleic acid.