Raman spectroscopic studies of the DNA cro binding site conformation, free and bound to cro protein

Raman spectra of the DNA binding site for cro repressor protein were obtained in the presence and absence of bound cro protein. The 17 base pair fragment is a consensus sequence of the six cro binding sites in phage lambda, except that the second base to the right of the center of pseudosymmetry is altered. Analysis of the spectrum of the free DNA indicates that the molecule exists in a B-like conformation with deviations from the usual B form occurring mainly in the bands assigned to A-T vibrations. The spectrum of the bound DNA was obtained by subtracting the spectrum of free cro from the spectrum of the complex which was estimated to be 90% bound. The DNA undergoes significant structural changes upon binding to the protein; most notable of these changes is a destacking of the G-C bases reflected by increases in the 1240, 1262, and 1320 cm-1 bands. A decrease in the 1361 cm-1 band that occurs has also been assigned to a destacking in guanine bases. The appearance of a 705 cm-1 band and the decrease and downshift of the 670 cm-1 band are consistent with the appearance of A-like character in the A-T region of the binding site when the protein binds; however, the spectra indicate that the entire binding site remains in a distorted B-like conformation. We use the 705 cm-1 band to estimate A-like character because the 800-850 cm-1 region is obscured by interference from strong protein bands. Other shifts in both intensity and position cannot be assigned to characteristic changes in conformation and therefore must be attributed to the protein influencing the structure in a novel way.