Estudio cualitativo para el servicio de tarifa mixta

6.8.1 Sedimentation assay

Using a sedimentation assay, we wanted to investigate whether the mutant YgfE-I83E can bind to FtsZ even though it does not induce FtsZ bundling, as concluded from light scattering. Through ultracentrifugation, the large polymers go down to the bottom, and can be separated to identify the proteins by SDS-PAGE.

A ratio of 1:2 for FtsZ and YgfE was chosen for the experiment, and control experiments with YgfE or FtsZ on their own were also performed. 800 μl reaction mixtures consisting of 22 μM of wild type YgfE, 22 μM of YgfE-I83E, 11 μM of FtsZ, 11 μM of FtsZ and 22 μM of wild type YgfE, 11μM of FtsZ and 22 μM of YgfE-I83E were prepared respectively in the polymerization buffer of 50 mM of MES-KOH, pH 6.5, 10 mM of MgCl2, and 50 mM of KCl. Immediately after

addition of GTP (2.5 mM), the centrifugation was performed at 80,000 RPM, 20°C for 10 minutes. In order to retain FtsZ in polymeric form throughout, we added a large excess of GTP, instead of 0.2 mM as normal. After that, 80μl of 50 mM Tris, pH 8, was used to resuspend each pellet. Samples from both supernatant and pellet were analysed by 15% SDS-PAGE.

The gel is shown in figure 6.8.1 labeled with supernatant (S) and pellet (P). When YgfE (either wild type or I83E) was mixed with GTP, there is no YgfE observed in the pellet (lanes 1 and 4), which means GTP can not bind to YgfE to form polymers. When FtsZ was mixed with GTP, FtsZ was observed in the pellet (lane 2P or lane 5P) indicates that FtsZ protofilaments were formed in the presence of GTP. Wild type YgfE helps to form FtsZ bundles, and both FtsZ and YgfE (lane 3P) can be seen in the pellet, when GTP was added into premixed FtsZ and YgfE. Similarly, we can find almost the same amount of both YgfE-I83E and FtsZ in the pellet (lane 6P) after the addition of GTP into premixed FtsZ and YgfE-I83E, which suggests that

the mutant YgfE-I83E did attached to FtsZ protofilaments, although it does not induce FtsZ bundling.

Figure 6.8.1 15% SDS-PAGE gel. M is marked for protein marker; S and P are marked for

supernatant and pellet respectively. Where present, FtsZ and YgfE were at 11μM and 22μM in polymerization buffer (50 mM MES-KOH, pH 6.5, 10 mM MgCl2and 50 mM KCl). (1) wild

type YgfE + GTP, (2) FtsZ + GTP, (3) FtsZ + wild type YgfE + GTP, (4) YgfE mutant I83E +

6.8.2 Dynamic light scattering

The dynamic light scattering was used to determine sizes of particles present in solution of mixtures of YgfE-I83E and FtsZ protofilaments. The particle size was measured using the DLS instrument Zetasizer Nano S (Malvern).

0 5 10 15 0 2 4 6 8 FtsZ+GTP FtsZ+YgfE-I83E+GTP FtsZ+YgfE (WT)+GTP M e a n V o lu m e (% ) Size (d. nm)

Figure 6.8.2 Volume particle size distributions obtained for different samples as indicated

measured on a Nano S instrument using backscatter detection. 0.4 mM of GTP was added into

the premixed solution to start the polymerization.

11 µM FtsZ and 44 µM of YgfE were premixed in the polymerization buffer consisting of 50 mM MES-KOH, pH 6.5, 10 mM MgCl2and 50 mM KCl. GTP to a

final concentration of 0.4 mM, instead of 0.2 mM as normal, was added to start the polymerization, which prolongs the steady state time of FtsZ polymerization. Immediately after the addition of GTP, the solution was transferred to the

DTS2145-low volume glass cuvette, and the DLS was observed at a 173° scattering angle.

The volume particle size distributions obtained from DLS are shown in figure 6.8.2. FtsZ protofilmants gave a peak around 1 nm diameter, whereas the peak changed significantly to around 2 nm in the presence of wild type YgfE, which revealed that wild type YgfE enhanced the polymerization of FtsZ. When GTP was added into the premixed solution containing FtsZ and the mutant YgfE-I83E, the DLS peak was shifted right a little bit. These data suggested that FtsZ bundles like those induced by wild type YgfE were not formed upon addition of YgfE-I83E. However, YgfE-I83E did cause the DLS peak to shift slightly, suggesting it binds to FtsZ protofilaments. Thus the results are in agreement with the previous sedimentation assay, although the maxima in the peak size distribution do not necessarily correspond to the precise values of the actual diameter of each species, because the FtsZ polymers are long fibres rather than spheres.

6.8.3 Probing whether YgfE-I83E blocks FtsZ bundling by

wild- type YgfE

Right-angle light scattering was continued to use to prove that the mutant YgfE-I83E is able to bind to FtsZ protofilments. 11 µM FtsZ and 44 µM of YgfE-I83E were premixed in the polymerization buffer consisting of 50 mM MES-KOH, pH 6.5, 10 mM MgCl2 and 50 mM KCl. After baseline collection of

approximately 5 minutes, GTP to a final concentration of 0.2 mM was added to initiate the polymerization, followed by the addition of 44 µM of wild type YgfE during the steady-state phase of the FtsZ polymerization, at approximately 3 minutes after the GTP addition.

0 100 200 300 400 500 600 0 200 400 600 800 1000 1200 1400 1:0 FtsZ:YgfE(WT) 1:4 [FtsZ+YgfE-I83E]:YgfE(WT) 1:4 FtsZ:YgfE(WT)

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Figure 6.8.3The block of FtsZ bundling by YgfE-I83E proved by right-angle light scattering.

11μM of FtsZ and 44μM of YgfE-I83E were premixed in the polymerization buffer, 44μM of wild type YgfE were added after FtsZ protofilament formation (approximately 3 minutes after

0.2 mM GTP addition at time zero). FtsZ polymerization on its own and adding wild type YgfE

into FtsZ protofilaments containing no YgfE-I83E were also shown for comparison. Excitation

and emission wavelengths were set at 550 nm.

Figure 6.8.3 shows the light scattering data obtained. After adding GTP into premixed FtsZ and YgfE-I83E, theres is a slight increase in the light scattering, which was identical to that by adding GTP into FtsZ alone, indicating only FtsZ protofilaments were formed. After that, wild type YgfE was added into FtsZ protofilaments containing YgfE-I83E, and a small increase in the light scattering (approximately 50 units) was observed, compared with the increase of 550 units that

was induced by adding wild type YgfE into FtsZ protofilaments on its own. Furthermore, this small increase in the light scattering rapidly decreased to zero within 4 minutes. The light scattering suggested that the wild type YgfE loses the abilty to induce FtsZ bundling in the presence of YgfE-I83E. This is presumably because YgfE-I83E binds to FtsZ protofilments and occupying the YgfE binding sites, therefore wild type YgfE is incapable of binding to FtsZ protofilments and thus induces the bundling.