Abstract
ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.
Highlights
RP437 [24]b RP5232 [24] RP5232 RP437 RP437 connected by a piece of polyethylene tubing (12 cm long, 0.58-mm inner diameter) [12]
All analysis of CyPet-MotB and of CyPet-FliN motors were performed using epifluorescence microscopy of fluorescent spots that were at the center of rotation of a tethered E. coli cell, ensuring that we analyzed only functioning motors
Our estimate of ~120 FliN molecules in wild-type motors is in very good agreement with our previous measurements of FliM copy numbers, assuming a 4:1 ratio (Fig. 2)
Summary
RP437 [24]b RP5232 [24] RP5232 RP437 RP437 connected by a piece of polyethylene tubing (12 cm long, 0.58-mm inner diameter) [12]. The cell suspension was centrifuged 3 times at 6,000 ϫ g for 3 min and resuspended in 75 l motility buffer. The cells were flowed through a tunnel slide and left to incubate for 5 min. Motility buffer containing 50 l·mlϪ1 of chloramphenicol was flushed through the tunnel slide to remove unbound cells. Laser epifluorescence illumination was used for all fluorescence imaging of the motor spots for measurements of the copy number of CyPet-FliN molecules, and total internal reflection fluorescence (TIRF) mode for complementation experiments. The intensities were ~0.127 W·mϪ2 and ~2 W·mϪ2 for the 440-nm (CyPet-MotB and CyPet-FliN) and 532-nm (FliM-YPet) illumination, respectively. Photobleaching of the CyPet-FliN motors was achieved by a 420-ms exposure to a focused laser spot (~3 mW·mϪ2) centered over the fluorescent spot at the center of rotation of the tethered cell. The total intensity integrated over the whole cell immediately after this photobleaching was 90% Ϯ 4% of that before photobleaching
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