Spectroscopic characterization of Co(II)-, Ni(II)-, and Cd(II)-substituted wild-type and non-native retroviral-type zinc finger peptides

摘要

The nucleocapsid protein (NCP) from Mason-Pfizer monkey virus (MPMV) contains two evolutionary invariant Cys-X_2-Cys-X_4-His-X_4-Cys retroviral-type zinc finger structures, where the Cys and His residues provide ligands to a tetrahedrally coordinated Zn(II) ion. The N-terminal zinc finger (F1) of NCP from MPMV contains an immediately contiguous Cys in the -1 position relative to the start of this conserved motif: Cys-Cys-X_2-Cys-X_4-His-X_4-Cys. Metal complexes of 18-amino acid peptides which model the native zinc finger sequence, SER-Cys-X_2-Cys-X_4-His-X_4-Cys (F1_SC), and non-native Cys-SER-X_2-Cys-X_4-His-X_4-Cys (F1_CS) and SER-SER-X_2-CYS-X_4-His-X_4-Cys (F1_SS) sequences have been spectroscopically characterized and compared to the native two-zinc-finger protein fragment, MPMV NCP 21-80. All Co(II)-substituted peptide complexes adopt tetrahedral ligand geometries and have S~-→Co(II) ligand-to-metal charge-transfer (LMCT) transition intensities consistent with three Co(II)-S bonds for F1_SC and F1_CS. The non-native F1_CS peptide binds Co(II) with K_(Co) = 1.5 * 10~6 M~(-1), comparable to that of the native complex, and ≈100-fold tighter than F1_SS. Like the Co(II) derivative, the absorption spectrum of Ni(II)-substituted NCP 21-80 is most consistent with tetrahedral Ni(II) complexes with multiple thiolate donors. In contrast, Ni(II) complexes of F1_SC and F1_CS exhibit a single absorption band in the 400-550 nm region (ε ≈ 200-300 M~(-1) cm~(-1)), distinct in the two complexes, assignable to a degenerate d-d transition envelope characteristic of non-native square-planar coordination geometry, and an intense LMCT transition in the UV (ε_(225) ≈ 14,000 M~(-1) cm~(-1)). Cd(II) complexes have intense absorption in the UV (λ_(max) = 233 nm), with absolute intensities consistent with ≈500 M~(-1) cm~(-1) per Cd(II)-S bond. ~(113)Cd NMR spectroscopy of ~(113)Cd MPMV NCP gives δ = 649 ppm, consistent with S_3N coordination. Co(II) and Cd(II) complexes of non-native F1_CS peptides are more sensitive to oxidation by O_2, relative to F1_SC, suggestive of a higher lability in the non-native chelate. The implications of these findings for the evolutionary conservation of this motif are discussed.