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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    • "where R 0 is the Fö rster distance for a particular donor/acceptor pair(Selvin, 1995; Taraska and Zagotta, 2007). Values of 16 A ˚ for F5M/Cu 2+ , 12 A ˚ for F5M/Ni 2+ , 12 A ˚ for mBBr/Cu 2+ and 10 A ˚ for mBBr/Ni 2+ were used (Taraska et al., 2009a, 2009b). "
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    ABSTRACT: Mapping the landscape of a protein's conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein maltose binding protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells.
    Structure 12/2012; 21(1). DOI:10.1016/j.str.2012.11.013 · 6.79 Impact Factor
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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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    ABSTRACT: A series of model dye-labeled histidine-containing peptides was used to investigate the nature of the quenching mechanism with Cu(2+) and Ni(2+). The strong reduction in steady-state fluorescence was found to be unaccompanied by any noticeable changes in lifetime kinetics. This static nature of quenching is not consistent with the dynamic Förster resonance energy transfer (FRET) phenomenon, which was assumed to dominate the quenching mechanism, and is likely caused by shorter range orbital coupling. Our results indicate that the FRET-like sixth power of distance dependence of quenching cannot be automatically assumed for transition metal ions and that time-resolved measurements should be used to distinguish various quenching mechanisms.
    Analytical Biochemistry 12/2010; 407(2):284-6. DOI:10.1016/j.ab.2010.07.035 · 2.22 Impact Factor
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    • "The attention to the investigation of dynamics of atoms, and especially collective dynamics of biomolecules and proteins, increased very much because of the importance of their biological functions [1] [2] [3]. Such informative methods as X-ray dynamical analysis, nuclear magnetic resonance, neutron scattering are used for these investigations [4]. "
    Lithuanian Journal of Physics 01/2010; DOI:10.3952/lithjphys.50411 · 0.46 Impact Factor
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