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Nat Methods. 2009 Jul;6(7):532-7. doi: 10.1038/nmeth.1341. Epub 2009 Jun 14.

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

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  • 1Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.

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.

PMID:
19525958
[PubMed - indexed for MEDLINE]
PMCID:
PMC2738593
Free PMC Article

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