Single-molecule FRET observations of conformational transitions of kinesin dimer with reduced neck linker tension. (A) Schematic depicting off-pathway transitions from the one-head–bound state to two-head–bound states of a neck linker extended mutant (5 poly-Gly insertion; green) without ATP. Positions of cysteine residues for labeling with the FRET probe (215Cys on the one head and 43Cys on the other head) are shown in orange spheres. (B) Typical FRET efficiency traces of Cy3/Cy5-labeled wild-type (G0) and neck linker extended mutants (3- and 5-Gly insertions; G3 and G5) bound to axonemes in the presence of 2 µM ADP. Blue and magenta dotted lines show previously reported peak FRET efficiencies for two-head–bound and one-head–bound states, respectively (Mori et al., 2007). (C) Histograms of FRET efficiencies (from each frame of images) of Cy3/Cy5-labeled kinesin with varied neck linker lengths bound to axonemes in the presence of 0.2, 2, or 10 µM ADP. Occasional ADP binding to kinesin in the two-head–bound state reverts to the one-head–bound state, and this frequency increases with higher ADP concentrations. Parentheses show the numbers of molecules analyzed. Dotted lines highlight peaks characteristic of putative two-head–bound (red) and one-head–bound (green) states.