FLUORESCENCE, PHOSPHORESCENCE, AND OPTICALLY DETECTED MAGNETIC-RESONANCE STUDIES OF THE NUCLEIC-ACID ASSOCIATION OF THE NUCLEOCAPSID PROTEIN OF THE MURINE LEUKEMIA-VIRUS

Fluorescence, phosphorescence, and optical detection of triplet state magnetic resonance (ODMR) are employed to investigate the interaction of p10, the nucleocapsid protein of the Moloney murine leukemia virus, with nucleic acids, p10 is a 55-amino acid protein containing a singl e zinc finger motif, (CCHC39)-C-26-H-29-C-34, that includes Y at posit ion 28 and W at position 35. In addition, the interactions of a zinc f inger peptide, p10-ZF, comprising residues 24-41 of p10, and a doubly mutated 24-41 peptide, p10-ZF' in which the positions of Y and W are i nterchanged, also are reported, The measurements focus on the direct i nvolvement of the sole W residue in the nucleic acid interaction. Fluo rescence quenching and salt-back titrations indicate complex formation of p10 with several octanucleotides-(dT)g, (dI)g, (dU)-dT, and (5-Brd U)(7)dT-and with the polynucleotides poly(dT) and poly(dI). Poly(dI) b inds with the highest affinity. Apparent binding constants and salt-ba ck midpoints are reported. Neither p10-ZF nor p10-ZF' exhibits signifi cant fluorescence quenching by these DNA substrates. Binding of p10-ZF to fluorescent poly(ethenoadenylic acid) was detected with greatly re duced affinity relative to p10, but binding of p10-ZF' was undetectabl e, These results are in general agreement with phosphorescence and ODM R measurements monitoring W, Addition of poly(I) to p10 leads to a pho sphorescence red shift, reduction in the zero-field splitting (ZFS) pa rameters D and E, and a significantly reduced phosphorescence lifetime , each consistent with aromatic stacking interactions between W and th e nucleobases. These effects are smaller with p10-ZF and undetectable with p10-ZF'. Poly(U) produces no significant changes in the triplet s tate parameters of W; no stacking interactions are observed even for p 10, (5-BrdU)(7)dT yields large phosphorescence red shifts in p10 and p 10-ZF, and reductions of D, but no significant heavy atom effects. The se effects probably are due to enhanced local polarizability caused by Br, but any stacking interactions in these complexes would exclude va n der Waals contacts between W and the Br atoms.