Article abstract
Nature Methods 6, 532 - 537 (2009)
Published online: 14 June 2009 | doi:10.1038/nmeth.1341
Mapping the structure and conformational movements of proteins with transition metal ion FRET
Justin W Taraska1, Michael C Puljung1, Nelson B Olivier2, Galen E Flynn1 & William N Zagotta1
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.
- Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Correspondence to: William N Zagotta1 e-mail: zagotta@u.washington.edu
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