Fig. 2: Conformations of ATL1cyto dimers in the presence of GTPγS and GDP/AlF4− revealed by intermolecular smFRET assays.
From: Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level
a As in Fig. 1a, but K400 in each protomer was selected for dye labeling and is represented as green and red spheres. b Strategy of the intermolecular smFRET assays for ATL1cyto-K dimers in the presence of GTPγS and GDP/AlF4−. The dimer is formed by a LD555-labeled ATL1cyto-K and LD655-labeled ATL1cyto-K. One protomer is biotinylated at the C-terminus. The biotin-streptavidin interaction was used to immobilize the protein in a streptavidin-coated microfluidic chamber. c Representative fluorescence and smFRET trajectories of intermolecular smFRET assays for ATL1cyto-K dimers in the presence of GTPγS (left and middle) and GDP/AlF4− (right). LD555 (the donor) is shown in green, LD655 (the acceptor) in red, and FRET in dark blue. d Intermolecular smFRET distributions of ATL1cyto-K dimers in the presence of GTPγS (left) and GDP/AlF4− (right). All of the individual FRET values with the number of molecules (Nm) displayed were compiled into a conformation-population FRET histogram (gray lines) and fitted into a two-state GaussAmp distribution (left, centered at ~0.28 in red line and ~0.66 in blue line) and one-state GaussAmp distribution (right, centered at ~0.66). Each bar height represents the normalized count (%). The length of the error bar represents the normalized SD of a Poissom distribution from the count. e The relative occupancy of the low-FRET state and high-FRET state for ATL1cyto-K dimers in the presence of GTPγS as derived from histograms. Data are presented as mean ± SD (n = 3 independent experiments) determined from three randomly assigned populations of all FRET traces. Source data are provided as a Source Data file.
