Min S. Lee

Relationship between1H and13C NMR chemical shifts and the secondary and tertiary structure of a zinc finger peptide

SummaryEssentially complete assignments have been obtained for the1H and protonated13C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the1H and13C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone CaH and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their1H chemical shifts and secondary structure.

Zinc is required for folding and binding of a single zinc finger to DNA

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non‐specifically to DNA only in the presence of zinc. Moreover, competive DNA binding experiments indicate interaction with 3.9 ± base pairs.

Complete assignment of the 1H NMR spectrum of a synthetic zinc finger from Xfin Sequential resonance assignments and secondary structure

A 25‐residue synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a compact, folded conformation in the presence of zinc. Complete 1H resonance assignments have been made. The peptide contains a helix, beginning as an α‐helix and ending as a 310‐helix, that extends from residue 12 to 23. Several positively charged and polar residues located on this helix are likely to be involved in interactions with DNA. Residues 1–10 appear to adopt a hairpin‐like structure.

The zinc finger motif. Conservation of chemical shifts and correlation with structure.

Zinc fingers of the transcription factor IIIA (TFIIIA) type, in which zinc is co‐ordinated by two cysteine and two histidine ligands (Cys2/His2), contain a length of helix packed against a β‐hairpin. These zinc fingers comprise the wider range of structurally homologous proteins for which 1H chemical shifts are available. A number of key resonances have chemical shifts that are highly sensitive to tertiary structure and are conserved between these peptides. The high conservation of these fingerprint chemical shifts is correlated with the common global fold of Cys2/His2 zinc fingers. These chemical shifts are largely independent of primary structure and should facilitate NMR assignments for future zinc finger proteins, as well as provide a diagnostic signature for the characteristic Cys2/His2 zinc finger fold.

Structural determinants of Cys2His2 zinc fingers

Two mutants of the zinc finger peptide Xfin‐31 (Ac‐YKCGLCERSFVEKSALSRHQRVHKN‐CONH2) containing alterations to the conserved hydrophobic core have been constructed and their zinc‐bound structures investigated by 1H NMR techniques. In the first (Xfin‐31B) a double mutation R8F/F10G places the conserved core aromatic residue at position 8 rather than position 10. In the second (Xfin‐31C), Phe‐10 is replaced by Leu. A qualitative analysis of 1H chemical shifts, NOE connectivities and coupling constants indicates that the global folds of both mutants are similar to that of the wild‐type protein. However, amide exchange rates suggest that the F10L mutant is much less stable than either the wild‐type or the R8F/F10G mutant.