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Mapping the structure and conformational movements of proteins with transition metal ion FRET

Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.
Nature Methods (Impact Factor: 25.95). 07/2009; 6(7):532-7. DOI: 10.1038/nmeth.1341
Source: PubMed

ABSTRACT Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

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    • ". Steady-state (A and B) and time-resolved (C and D) quenching of fluorescein-labeled (A and C) and bimane-labeled (B and D) model peptide C2H6H10 using transition metal ions. Steady-state fluorescence titration with Cu 2+ (squares) and Ni 2+ (circles) results in substantial reduction of intensity (open symbols are published data of Taraska and coworkers [2] [3], and closed symbols represent our measurements). Both time-resolved fluorescence decays (C and D) and steady-state intensities (A and B) were measured for the following: samples containing no quencher (F1 and B1) and samples with saturating concentrations of Ni 2+ (F2 and B2) and Cu 2+ (F3 and B3). "
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