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

Nature Methods volume 6, pages 532–537 (2009)Cite this article

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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Figure 1: Measuring distances with transition metal ion FRET.
Figure 2: Mapping distances in HCN2 bound to cAMP with transition metal ion FRET.
Figure 3: Crystal structure of the mouse wild-type HCN2 C-terminal fragment (C-linker and CNBD) in the absence of cAMP.
Figure 4: Structural rearrangements in HCN2 determined with transition metal ion FRET.
Figure 5: The C-helix is stabilized during cAMP-binding.

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Acknowledgements

We thank K. Black, S. Cunnington, S. Simmons and G. Sheridan for technical assistance, the beamline staff at the Advanced Light Source and National Synchrotron Light Source for help with data collection, W. Almers, B. Hille and A. Merz for comments on the manuscript, and E. Gouaux and L. Islas for stimulating discussions. This work was supported by the Howard Hughes Medical Institute and National Institutes of Health (NIH) grants EY10329 to W.N.Z. and R01 NS038631 to E. Gouaux. M.C.P was supported by a Ruth Kirschstein National Research Service Award from the National Eye Institute. J.W.T was supported by a fellowship from the Jane Coffin Childs Foundation and a NIH Pathway to Independence Award (1K99NS064213).

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Authors and Affiliations

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

    Justin W Taraska, Michael C Puljung, Galen E Flynn & William N Zagotta

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Nelson B Olivier

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  1. Justin W Taraska
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  2. Michael C Puljung
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  3. Nelson B Olivier
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  4. Galen E Flynn
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Contributions

J.W.T designed the experiments and analysis. J.W.T and M.C.P. performed fluorescence experiments. N.B.O, G.E.F. and W.N.Z. performed the crystallography. J.W.T and W.N.Z. wrote the manuscript.

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Correspondence to William N Zagotta.

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Supplementary Figures 1–7, Supplementary Tables 1–2 (PDF 2096 kb)

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Taraska, J., Puljung, M., Olivier, N. et al. Mapping the structure and conformational movements of proteins with transition metal ion FRET. Nat Methods 6, 532–537 (2009). https://doi.org/10.1038/nmeth.1341

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