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 single zinc finger motif, C26C29H34C39, that includes Y at position 28 and W at position 35. In addition, the interactions of a zinc finger 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 interchanged, also are reported. The measurements focus on the direct involvement of the sole W residue in the nucleic acid interaction. Fluorescence quenching and salt-back titrations indicate complex formation of p10 with several octanucleotides--(dT)8, (dI)8, (dU)7dT, and (5-BrdU)7dT--and with the polynucleotides poly(dT) and poly(dI). Poly(dI) binds with the highest affinity. Apparent binding constants and salt-back midpoints are reported. Neither p10-ZF nor p10-ZF' exhibits significant fluorescence quenching by these DNA substrates. Binding of p10-ZF to fluorescent poly(ethenoadenylic acid) was detected with greatly reduced affinity relative to p10, but binding of p10-ZF' was undetectable. These results are in general agreement with phosphorescence and ODMR measurements monitoring W. Addition of poly(I) to p10 leads to a phosphorescence red shift, reduction in the zero-field splitting (ZFS) parameters D and E, and a significantly reduced phosphorescence lifetime, each consistent with aromatic stacking interactions between W and the nucleobases. These effects are smaller with p10-ZF and undetectable with p10-ZF'. Poly(U) produces no significant changes in the triplet state parameters of W; no stacking interactions are observed even for p10. (5-BrdU)7dT yields large phosphorescence red shifts in p10 and p10-ZF, and reductions of D, but no significant heavy atom effects. These effects probably are due to enhanced local polarizability caused by Br, but any stacking interactions in these complexes would exclude van der Waals contacts between W and the Br atoms.
Wu
J Q
JQ
Department of Chemistry, University of California, Davis 95616, USA.
Maki
A H
AH
Ozarowski
A
A
Urbaneja
M A
MA
Henderson
L E
LE
Casas-Finet
J R
JR
D000595:Amino Acid Sequence; D015683:Gene Products, gag / Q000378:metabolism*; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011069:Poly I / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*; D016335:Zinc Fingers
D015683:Gene Products, gag; D009838:Oligodeoxyribonucleotides; D014758:Viral Core Proteins; C068662:retroviral nucleocapsid protein NCp10, Moloney murine leukemia virus; D011069:Poly I; D014364:Tryptophan