Spin-Labeled Nucleotide Mobility in the Boundary of theEcoRI Endonuclease Binding Site

Abstract

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 binding. 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 region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized 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 well 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 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results 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 DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated 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 hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.

Mapping RNA−Protein Interactions in Ribonuclease P from Escherichia coli Using Electron Paramagnetic Resonance Spectroscopy

“…Results similar to those obtained in this study have been observed in earlier investigations wherein spin-labeled nucleic acid molecules were used to study the structure of nucleoprotein complexes (8,9,39). While studying by EPR spectroscopy the encapsidation of spin-labeled poly(A) by tobacco mosaic virus (TMV) protein to form viral-like particles, a pronounced broadening of the EPR spectra was observed during in vitro assembly of the viral-like particle (8).…”

“…Another study examining the binding of bacteriophage fd gene 5 protein to spin-labeled polynucleotides revealed a significant change in the EPR line shape upon formation of the DNA-protein complex (9). Finally, a complex of EcoRI bound to spin-labeled DNA oligonucleotides (containing the recognition site for EcoRI) was investigated by EPR spectroscopy to determine the conformational changes in the DNA that occur upon binding to EcoRI (39). The changes in spectral line shapes were used to infer that a spin label positioned proximal to the GAATTC recognition site is indeed influenced by the binding event (39).…”

A Distance Ruler for RNA Using EPR and Site-Directed Spin Labeling

“…In the case of proteins, molecules, and in order to apply this method to predict thiol-reactive nitroxide spin labels have been introduced the folding pathways of larger RNA molecules, six difmainly to cysteine-substituted sites [2][3][4][5]. For RNA oliferent short 10-mer RNA duplexes have been designed gonucleotides, spin labels can be covalently attached with spin labels attached at varying 2Ј positions (Figeither to modified bases [6][7][8] or the phosphate moiety ure 1) such that the interspin distances vary between [9, 10], or to the 2Ј position of the ribose ring [11,12].…”

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

“…The first line of work in-volves monitoring the dynamics of protein-nucleic acid complexes. This is accomplished by placing spin labels either inside the protein binding site, as with polylysine (Bobst et al, 1985) and gene 5 single-strand binding protein , or outside the binding site, as in the cases of the site-specific EcoRl endonuclease (Keyes et al, 1996) and the homeodomain (Bobst et al, 1996b). By placing the labels at various distances from the binding site, the transmission of structural information can be monitored.…”