The behaviors of a monofunctional fluorophore–attached helix 6A. (A) The intensities recorded from a monofunctional fluorophore attached to an AdiC molecule in the absence of any substrate. Individual components are color coded and plotted against the observation time. Each black vertical line in the black traces, superimposed on the colored intensity traces, indicates the time point at which a change in the intensities and thus fluorophore’s orientation is identified, whereas each black horizontal line represents the mean intensity between two identified consecutive time points. (B) Traces for θ and φ are calculated from the black traces in A and expressed in the local frame of reference defined in Results. Values for the Ω trace are calculated from the θ and φ traces relative to the mean values for C1, where the states are color coded. (C) The 3D probability densities of individual conformational states plotted against θ and φ values. The θ and φ values for individual events are determined from the particle exhibited in panel A. (D) The positions of the arrowheads of individual vectors that represents the orientations of the fluorophore dipole in the individual events of adopting various conformations are mapped onto a unit sphere defined by the Cartesian coordinates of the local framework described in Results. The x, y, and z positions are calculated from θ and φ plotted in panel C, with a radius of a unit length that carry no physical meaning here. (E and F) Ensemble 3D probability density distributions of θ and φ in the absence (apo) and the presence of 1 mM Arg+. The distributions were built with the data analyzed from 25 to 36 particles with a total of 584–699 events. The histograms (C, E, and F) and data points (D) for conformational state C1 are colored yellow, C2 colored blue, C3 colored orange, and C4 color cyan.
