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, 88 (1), 505-14

A Rapid Fluorescence Assay for FtsZ Assembly Indicates Cooperative Assembly With a Dimer Nucleus

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A Rapid Fluorescence Assay for FtsZ Assembly Indicates Cooperative Assembly With a Dimer Nucleus

Yaodong Chen et al. Biophys J.

Abstract

FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?

Figures

FIGURE 1
FIGURE 1
The tryptophan fluorescence emission spectrum of 0.5 μM FtsZ L68W in MMK buffer, excited at 295 nm, is shown before and after addition of 0.2 mM GDP and GTP.
FIGURE 2
FIGURE 2
Fluorescence of L68W as a function of protein concentration, in HEK buffer, pH 7.7. (a) Fluorescence is shown for FtsZ before and 150 s after addition of GTP. (b) The no-GTP curve was subtracted from the GTP curve to show the fluorescence enhancement, which is interpreted to be the measure of total polymer. The CC in this buffer, with no Mg, is 0.4 μM.
FIGURE 3
FIGURE 3
Assembly of L68W in HMK buffer, pH 7.7. The fluorescence for FtsZ-L68W before and 150 s after addition of GTP are shown. In this buffer, which contains Mg and permits GTP hydrolysis, the CC was reduced to 0.1 μM.
FIGURE 4
FIGURE 4
Assembly in HE buffer, pH 7.7, with variable concentrations of KAc. Assembly appeared to be identical in 100 and 350 mM KAc, with CC = 0.45 μM. In the absence of potassium the CC increased dramatically to 2.77 μM, and the slope of the curve above CC also decreased.
FIGURE 5
FIGURE 5
EM of FtsZ-L68W assembled at 1 μM protein for two min, in HEK and MEK buffers. The protofilaments appear to be one-subunit thick. They show a tendency to curve, forming circles ∼200–300 nm in diameter. (This is quite different from the curved conformation, which forms rings and tubes of 23-nm diameter.)
FIGURE 6
FIGURE 6
Stopped-flow data (circles) and the best fit from KINSIM (lines) for assembly in different buffers. The insets in c and d show the lower concentrations on an expanded scale. The concentration of FtsZ is indicated by each curve.
FIGURE 7
FIGURE 7
The model used to fit the stopped-flow kinetic data. The first step is monomer activation, probably dissociation of the bound GDP. This is followed by formation of a weak dimer nucleus, and then by steps of elongation. The model shows the polymer as a two-stranded filament because this is the simplest explanation for a dimer nucleus, but this is contradicted by the EM, which shows FtsZ protofilaments to be single-stranded.

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