title: Time-resolved energy transfer measurements of donor-acceptor distance distributions and intramolecular flexibility of a CCHH zinc finger peptide.
abstract: Time-resolved frequency-domain fluorescence energy transfer measurements have been used to investigate the solution structure of a single-domain CCHH-type zinc finger peptide. These measurements reveal not only the range of accessible distances for a given donor-acceptor pair within the molecule but also the degree of conformational flexibility that occurs in solution. Two donor-acceptor (D-A)-pair zinc finger peptides have been synthesized. A single tryptophan residue located at the midpoint of the sequence was the energy donor for two different acceptors. One acceptor, attached at the amino terminus was a 5-(dimethylamino)-1-naphthalenesulfonyl (DNS) group; the second acceptor was a 7-amino-4-methyl-coumarin-3-acetyl (AMCA) group, attached to the epsilon-amino function of a carboxy-terminal lysine residue. Distance distributions and the mutual site-to-site diffusion coefficients were determined for these two D-A-labeled peptides under zinc-bound, metal-free, and denatured conditions. The D-A distance distributions determined for these two peptides under metal-free and zinc-bound conditions 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. No site-to-site diffusion was detected for the zinc-bound peptide, whereas an appreciable amount of diffusion was measured for both metal-free and denatured peptide. Anisotropy measurements on the peptides indicated increased flexibility of all regions of the peptide chain in the absence of zinc and a more compact, less flexible structure when zinc was bound. It was concluded from these results that the metal-bound conformation represents a unique, well-defined structure. Comparison of distance distributions measured for metal-free and denatured peptide indicated that there is some residual structure present in the metal-free peptide.
authors.affiliation: Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201.
authors.forename: P S
authors.initials: PS
authors.lastname: Eis
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004735:Energy Transfer; D005454:Fluorescence Polarization; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins