DINUCLEOTIDE BINDING TO RNASE PROBED WITH UV RESONANCE RAMAN-SPECTROSCOPY

Raman spectra are reported for ribonuclease and its complexes with the dideoxynucleotides dCpdC, dCpdA and dCpdG, using 240-250-nm excitation to enhance the purine and pyrimidine modes, as well as tyrosine and phenylalanine modes of the protein. Spectral perturbations associated with binding were examined via difference spectroscopy. Consistent with the crystal structure of CpA* bound to RNase, the 3'-cytidine was inferred to be hydrogen bonded to a protein donor via the N-3 ring atom, but not via the carbonyl O atom, since the 1526 cm-1 band shifts up on binding, as it does when aqueous CMP is protonated, and the C = O stretch shifts from 1655 to 1670 cm-1, an effect attributable to polarization via the N-3 hydrogen bond. The effect of binding on the spectra of the 5'-purine rings is limited to intensity changes, an augmentation of the adenine 1578 cm-1 band and a diminution of the guanine 1330 cm-1 band, consistent with only weak interactions with the protein. Binding of dCpdC intensifies the 1615 cm-1 protein band, suggesting environmental changes for tyrosine or phenylalanine residues. No spectral perturbation was observed on mixing RNase with dGpdC or dGpdG, consistent with weak or non-specific binding of dinucleotides lacking a pyrimidine in the 3'-position.