Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid.
The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.
Secnik
J
J
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Wang
Q
Q
Chang
C M
CM
Jentoft
J E
JE
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites / Q000187:drug effects; D002213:Capsid / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000378:metabolism; D011061:Poly A / Q000494:pharmacology; D012191:Retroviridae Proteins / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D012191:Retroviridae Proteins; D014758:Viral Core Proteins; C040520:avian retrovirus proteins; D011061:Poly A; D012965:Sodium Chloride; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)