Distance distributions and dynamics of a zinc finger peptide from fluorescence resonance energy transfer measurements.

Time-resolved fluorescence resonance energy transfer (FRET) measurements were used to measure distance distributions and intramolecular dynamics (site-to-site diffusion) of a 28-residue single-domain zinc finger peptide in the absence and presence of zinc ion. Energy transfer was measured between TRP14 and a N-terminal DNS group. As expected, the TRP-to-DNS distance distribution for zinc-bound peptide is shorter and narrower (R av=11.2 Å,hw=2.8 Å) than the metal-free peptide (R av=20.1 Å,hw=14.5 Å). The degree of mutual donor-to-acceptor diffusion (D) was also determined for these distributions. For zinc-bound peptide there is no detectible diffusion (D≤0.2 Å(2)/ns), whereas for metal-free peptide a considerable amount of motion is occurring between the donor and the acceptor (D=12 Å(2)/ns). These results indicate that the zinc-bound peptide folds into a unique, well-defined conformation, whereas the metal-free conformation is flexible and rapidly changing. The absence of detectible mutual site-to-site diffusion between the donor and the acceptor in the metal-bound zinc finger peptide indicates that intramolecular motion is essentially frozen out, on the FRET time scale, as a consequence of zinc coordination.