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


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.

19 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "In this variation of tmFRET (Taraska et al., 2009), quenching is determined by the distance between a TAMRA dye attached to K330C in the small AAA+ domain and a Ni 2+ ion bound to an a-helical His-X 3 -His motif in the large domain of the same subunit (Figure 4A). The His-X 3 -His site was engineered by introducing N72H and D76H mutations in combination with H68Q to remove an alternative Ni 2+ -binding site, and nitrilotriacetic acid (NTA) was included in assays to minimize Ni 2+ binding to nucleotides. "
    [Show abstract] [Hide abstract]
    ABSTRACT: ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.
    Cell 04/2013; 153(3):628-39. DOI:10.1016/j.cell.2013.03.029 · 32.24 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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
Show more

Preview (2 Sources)

19 Reads
Available from