Influence of the N- and C-terminal chains on the zinc-binding and conformational properties of the central zinc-finger structure of Moloney murine leukaemia virus nucleocapsid protein: a steady-state and time-resolved fluorescence study.
The nucleocapsid protein NCp10 of the Moloney murine leukaemia virus is a small basic protein characterized by a central Cys26-X2-Cys29-X4-His34-X4-Cys39 zinc-finger domain. Mutants with deletion of either the N- or C-terminal chain (or both) surrounding the central zinc-finger domain were synthesized by a solid-phase approach in order to evaluate the influence of these lateral chains on zinc binding and conformational properties of NCp10. For this purpose, the steady-state and time-resolved fluorescence properties of the single Trp-35 residue of the various NCp10 derivatives were analyzed. The binding properties of the various derivatives suggest that the central zinc-finger domain affinity for zinc is not modified by the N-terminal chain and is only slightly (about one order of magnitude) increased by the C-terminal chain leading to a Kapp of (1.2 +/- 0.2).10(14) M-1 for the whole NCp10. Concerning the conformation of the NCp10 derivatives, fluorescence data are in agreement with structureless polypeptide chains in the absence of zinc. In contrast, in the presence of zinc, the fluorescence intensity decays are in agreement with a unique conformation of the finger motif backbone and a distribution of the Trp-indole moiety into two classes with different local environments. Decay-associated spectra, fluorescence quenching by acrylamide and anisotropy decay data further suggest that the Trp-indole moiety of both classes was highly exposed to solvent and had a high degree of rotational freedom. Finally, in contrast to the C-terminal chain, the N-terminal chain modifies the local environment and the accessibility to external quenchers of both Trp-35 classes, suggesting that it was folded in the vicinity of the Trp-35 residue.
Mely
Y
Y
Laboratoire de Biophysique de la Faculté de Pharmacie, CNRS UA 491, Université Louis Pasteur, Strasbourg, Illkirch, France.
de Rocquigny
H
H
Piémont
E
E
Déméné
H
H
Jullian
N
N
Fournié-Zaluski
M C
MC
Roques
B
B
Gérard
D
D
D000595:Amino Acid Sequence; D015200:Fluoroimmunoassay; D015683:Gene Products, gag / Q000737:chemistry*; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000737:chemistry*; D011487:Protein Conformation; D014357:Trypsin; D014758:Viral Core Proteins / Q000737:chemistry*; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D015683:Gene Products, gag; D014758:Viral Core Proteins; C068662:retroviral nucleocapsid protein NCp10, Moloney murine leukemia virus; D014357:Trypsin; D015032:Zinc