Actividades de Mantenimiento basado en RCM Abreviado (GARCÍA, 2003)

As shown in Figure 5.5.1. the wild-type void fraction consists of a wide variety of irregular amorphous particles that vary greatly in both shape and size; particles ranging from ~8 nm to over 100 nm are evident. The smallest of these particles form a background of round and relatively homogenous structures. As evident from the biochemical data shown earlier (Figure 5.4.6.) this fraction consists of TatCy-his with no detectable TatAy or contaminants. Therefore it can be concluded that TatCy- his is able coalesce into a wide range of shapes and sizes in the absence of sufficient TatAy and that such complexes are structurally unstable at least under these purification conditions. If such complexes were stable all visible particles present in this gel filtration fraction would be expected to be over the 600 kDa size limit of the Superdex 200 column.

Figure 5.5.2. shows a 1:4 dilution of this fraction, prepared using gel filtration buffer + 0.02% DDM. Although the relative number of particles has been reduced the size distribution and morphology of the particles remains unaffected, showing that the large aggregates are not caused by overcrowding of the grid and that they cannot be disaggregated by simple dilution.

To provide further confirmation as to the constitution of these aggregates a grid was prepared with the addition of 1.8 nm Ni-NTA-Nanogold® (Nanoprobes). A fresh grid was prepared using the same 1:4 dilution of TatAyCy but the 1 minute incubation of the sample on the grid was followed by a 10 minute incubation with a 1:10 dilution of Nanogold (diluted in GF buffer minus detergent), before thoroughly washing and

Chapter 5: Structural investigation of TatAyCy complexes

169 staining (see methods). From Figure 5.5.3.A multiple intense black dots of 1.8 nm diameter can clearly be seen clustering over the large aggregates indicating that multiple his-tagged proteins are surface accessible. The Nanogold can be seen more clearly in the inset images shown in Figure 5.5.3.B+C. These results provided clear evidence that the aggregates consist of TatCy-his.

Figure 5.5.1. Micrograph of wild-type TatAyCy-his void fraction undiluted The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. TatAyCy forms a wide variety of irregular amorphous particles that vary greatly in both size and shape (~8 nm to over 100 nm).

Chapter 5: Structural investigation of TatAyCy complexes

170 Figure 5.5.2. Micrograph of wild-type TatAyCy-his void fraction1:4 dilution The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. TatAyCy forms a wide variety of irregular amorphous particles that vary greatly in both size and shape (~8 nm to over 100 nm).

Chapter 5: Structural investigation of TatAyCy complexes

171 Figure 5.5.3. Micrograph of wild-type TatAyCy-his void fraction+ Nanogold A. The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. Aggregates of TatCy-his extensively labelled with 1.8 µm Ni-NTA-Nanogold are indicated by arrows. B+C. In each case the left hand-panel shows a close-up view of one of the aggregates, and the right- hand panel has been processed using the despeckle tool of ImageJ (ver. 1.44p) to reduce the carbon grain and enhance the Nanogold signal.

B

100 nm

C

Chapter 5: Structural investigation of TatAyCy complexes

172 5.5.2. Single-particle EM of P2A mutant TatAyCy-his void fraction

Images taken of the corresponding void fraction for the mutant sample display a remarkably different phenotype to the wild-type as shown in Figure 5.5.4.A. An abundance of long tubular structures can be clearly seen in the undiluted sample. These are ~ 10 nm thick and range widely in length with an average spread of ~ 50- 200 nm. The tubules consist of repeating units each ~ 5.5 nm long with a diagonal arrangement as highlighted in Figure 5.5.4.B and Figure 5.5.4.C. Globular particles are also present in the sample with both large, amorphous particles (up to 50 nm) and small, round and homogenous ones seen.

Chapter 5: Structural investigation of TatAyCy complexes

173 Figure 5.5.4. Micrograph of (P2A) TatAyCy-his void fractionundiluted

A. The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. B. Close-up of micrograph section indicated C. A clearly defined tubule is shown in detail. From left to right: unprocessed image, image processed with 2.0 pixel radius Gaussian filter, image processed with 4.0 pixel radius Gaussian filter, schematic representation of helical structure of tubule. Processed using the Gaussian filter tool of ImageJ (ver. 1.44p).

This diverse range of particles was not altered upon a 1:8 dilution as seen in Figure 5.5.5. Again, this suggests that the observed particle populations are not simply caused, or affected, by protein concentration. Since this fraction contains an excess of TatAy and no significant contaminants, as shown by the protein biochemistry (see Figure 5.4.7.) it can be inferred that these tubules consist primarily, if not entirely, of the mutated TatAy. Furthermore, the repeating units of these tubules fit closely to the dimensions of other TatA-type complexes observed previously (see TatAd in chapter 3, and TatE in chapter 4), suggesting that the tubules are formed by polymerisation of smaller ‘ringed’ TatAy complexes. Such tubule formation by E. coli TatA has been observed previously in vivo within the cytoplasm of TatA over-expressing cells (Berthelmann et al., 2008). The large amorphous particles seen in this fraction closely match the morphology of those prevalent in the wild-type sample (Figure 5.5.1. to Figure 5.5.3.) suggesting that they are aggregates of, or at least contain, TatCy-his. The smaller ringed particles, shown more clearly in the diluted sample (Figure 5.5.5.), are also seen in the wild-type (Figure 5.5.2.) suggesting that these

B

20 nm 100 nm

Chapter 5: Structural investigation of TatAyCy complexes

174 constitute some form of TatAyCy-his complex with an unknown and potentially variable stoichiometry.

Nanogold was used again to further investigate the distribution of subunits within these particle populations. Figure 5.5.6. shows a 1:8 dilution of the mutant sample with bound 1.8 µm Ni-NTA-Nanogold® prepared in the same way as for the wild- type sample. Nanogold particles appear to be bound to the large amorphous particles and some of the smaller round particles, but not to the lengths of the tubules. Some nanogold is seen close to the extremities of tubules or to larger nodes present along their length, suggesting that residual TatCy-his may be present at these locations. Overall this evidence supports the theory that the tubules do not contain TatCy-his whilst the other particle populations do.

Chapter 5: Structural investigation of TatAyCy complexes

175 Figure 5.5.5. Micrograph of (P2A) TatAyCy-his void fraction,1:8 dilution The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate.

Chapter 5: Structural investigation of TatAyCy complexes

176 Figure 5.5.6. Micrograph of (P2A) TatAyCy-his void fraction+ Nanogold

A. The micrograph was taken at ~57,000x magnification under ~0.5 µm defocus. The grid was stained with 2% uranyl acetate. Several clusters of Nanogold particles are indicated by arrows. B+C. Inset of areas indicated in A. 2.0 pixel Gaussian filter applied. Processed using the Gaussian filter tool of ImageJ (ver. 1.44p).

B

C

A

Chapter 5: Structural investigation of TatAyCy complexes

177 To further investigate the structure these tubules in more detail, cryo-EM grids were prepared of the undiluted sample using the method discussed previously. Over 100 Images were taken at 71565x magnification using FasTEM minimal dose settings (MDS) at a range of defoci. As these tubules present a larger repeating structure than smaller Tat complexes they are more suitable for imaging under the very low contrast conditions of cryo-EM. In Figure 5.5.7.A and Figure 5.5.7.B multiple tubules can be seen adopting a variety of condensed and extended conformations, the longest of these (see Figure 5.5.7.B) measuring ~ 800 nm. These data confirm that these tubules are not an artefact introduced by the harsh conditions of negative-stain; the staining and blotting procedure induces aggregation and flattening effects on the protein as well as potentially restricting the orientation of the particles on the carbon surface. Under cryo-EM conditions such effects are mitigated and the sample is imaged in a pseudo-aqueous state. These images provide high resolution data for future single particle analysis of the tubules using a filamentous particle reconstruction technique.

Chapter 5: Structural investigation of TatAyCy complexes

178

Chapter 5: Structural investigation of TatAyCy complexes

179 Figure 5.5.7. Cryo-EM micrographs of (P2A) TatAyCy-his void fraction

A.+B. Micrographs were taken at ~72,000x magnification under minimal dose

settings. Samples were snap-frozen in liquid ethane to form vitreous ice. Several tubules are indicated by arrows.