Investigation of the solution structure of a zinc finger peptide with time-resolved fluorescence spectroscopy

Abstract

Zinc finger proteins are a class of nucleic acid binding proteins that tetrahedrally coordinate zinc ion via cysteine (C) and histidine (H) sidechain atoms to form a defined structure. A solution stucture of the CCHH type zinc finger has been determined with 2D NMR and an X-ray crystallographic structure of a zinc finger protein-DNA complex has been solved. However, neither of these methods reveal information about the conformational heterogeneity that exists in a zinc finger domain. Therefore, the solution structure of a single zinc finger peptide of the CCHH type was investigated with fluorescence spectroscopy in the absence and presence of metal ion and in a denatured state. Energy transfer distance distribution measurements were performed on two different donor-acceptor labeled peptides to determine the range of intramolecular distances and the degree of conformational heterogeneity that exists for the peptide in the metal-free and metal-bound state. Anisotropy measurements were also performed to assess the structure of the zinc finger peptide. A single tryptophan, located at the midpoint of the peptide sequence, serves as the energy donor for two different acceptors. One peptide contains a DNS acceptor attached at its amino terminus and another peptide contains a coumarin derivative attached at the {dollar}\epsilon{dollar}-amino group of its carboxy-terminal lysine. The donor-acceptor distance distributions determined for these two peptides in both cases indicated a shorter distance and a unique conformation (narrow distribution) when metal was bound, and a longer distance with greater conformational flexibility when metal ion was absent. Clearly the metal-bound conformation represents a unique, well-defined structure. Comparison of distance distributions measured for metal-free and denatured peptide indicates that there is some residual structure present in the metal-free peptide.