CW and CCW Conformations of the E CW and CCW Conformations of the E. coli Flagellar Motor C-Ring Evaluated by Fluorescence Anisotropy  Basarab G. Hosu, Howard C. Berg  Biophysical Journal  Volume 114, Issue 3, Pages 641-649 (February 2018) DOI: 10.1016/j.bpj.2017.12.001 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Experimental setup. The circuits used to control the deflection of the laser beam, the modulation of its phase, and the timing of the camera are not shown. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 (A) Fluorescence emission versus time (camera frame #) for free YFP expressed in the cytoplasm (green lines), CW motors labeled with FliN-YFPINT (blue lines), CW motors labeled with FliM-YFP (red lines), and a 10 μM solution of fluorescein (black lines). The samples were excited at 900 frames per second with light polarized in S for 50 μs per frame, and the emission was recorded in S (Ipar, solid lines) and P (Iper, dashed lines). (B) Normalized total fluorescence emission (I=Ipar+2⋅Iper) computed from the curves shown in (A). (C) Corresponding FA values, R = (Ipar − Iper)/I, versus time for free YFP (green), FliN-YFPINT (blue), FliM-YFP (red), and 10 μM fluorescein (black). The error bars, shown only for the first three data points in each curve, are standard errors. To see this figure in color, go online. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 FA versus time of motors labeled with FliN-YFPINT (A and B) or FliM-YFP (C and D), in strains that rotate steadily CW (blue lines) or CCW (red lines). The samples were excited at 900 frames/s with light polarized in S for 50 μs (A and C) or 100 μs (B and D). The error bars (shown for the first three data points in each curve only) are standard errors. To see this figure in color, go online. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Bootstrap results: FA versus total fluorescence emission of motors rotating CW (blue dots) or CCW (red dots) labeled with FliN-YFPINT (A and B) or FliM-YFP (C and D). The fluorescence emission in each panel is normalized to the mean CW value. The dots represent the average of the first three data points in each corresponding FA versus time curve shown in Fig 3. The samples were excited for 50 μs (A and C) or 100 μs (B and D) per camera frame. To see this figure in color, go online. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 FliN-YFPINT construct and possible homo-FRET pairs. (A) Ribbon representations of the resolved portion of a FliN monomer and a green fluorescent protein (very similar to YFP, left) superimposed to a cartoon representation of a FliN monomer (middle). The highly conserved, resolved section (amino acid residues 51–137) is red, the variable, not resolved section is blue, and YFP is yellow. The two N-terminal ends of the saddle-shaped dimer (right), one dimer colored as described, the other one gray, are hypothesized to brace each other over the opposite dimer (1), not represented. At the bottom, two possible configurations of the FliN-YFPINT tetramer (one FliN-YFPINT monomer color-coded as above, the other three in different shades of gray), with fluorophores spaced equally (B) or unequally (C). The curved lines with arrowheads at both ends depict the possible homo-FRET interactions. (B) Homo-FRET is equally likely to occur in between any pair of adjacent fluorophores within an isolated tetramer (δ ≥ 6 nm), but less likely when the tetramer is part of the C-ring, when intertetramer homo-FRET is more probable (δ = 4 nm, see text). In (C), homo-FRET is more likely to occur in between pairs of fluorophores on opposite dimers. Given the insertion location of YFP within the FliN monomer, this latter arrangement is more probable. To see this figure in color, go online. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Possible CW/CCW conformational change of FliN tetramers involving puckering of FliN-YFP dimers in opposite directions, accompanied by FliM rotation. Top: side view of the C-ring (left) at the cytoplasmic end of the motor, with an enlarged FliM monomer and a FliN-YFPINT tetramer in CW (middle) and CCW (right) conformations. Bottom: top view of the C-ring, with an interlaced arrangement of FliM monomers and FliN tetramers (left); an enlarged FliM monomer and two adjacent FliN-YFPINT tetramers are puckered in CW (middle) or CCW (right) conformations. CheY-P (not represented, in CW conformation) only and FliM hinder the puckering of the FliN-YFPINT tetramer at one of the two dimer-dimer interaction sites labeled with YFP1 and YFP3, but not at the other, labeled with YFP2 and YFP4. One dimer is composed by the monomers FliN1 and FliN2, labeled with YFP1 and FliN2, respectively; the other dimer is composed by the monomers FliN3 and FliN4, labeled with YFP3 and YFP4, respectively. Biophysical Journal 2018 114, 641-649DOI: (10.1016/j.bpj.2017.12.001) Copyright © 2017 Biophysical Society Terms and Conditions