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: 32.07). 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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    • "The residue in HCN2 (F518) that aligns with F525 in CNGB3 resides in the F’ helix and appears to be buried in this C-linker closed conformation [68]. The F’ helix of HCN2, in concert with the C helix of the CNBD, has been shown recently to be part of a key conformational rearrangement that occurs upon ligand binding and is proposed to help propagate the gating transition through the C-linker region to the pore [71]. The phenylalanine to asparagine substitution in CNGB3 reduces both side-chain volume and hydrophobicity. "
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    ABSTRACT: To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.
    Molecular vision 06/2013; 19:1268-1281. · 1.99 Impact Factor
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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 · 5.62 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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