Samples were taken from gel filtration fractions covering the peak of Tat protein elution (22-26). Initial negative stain grids were prepared using undiluted aliquots to avoid the potential introduction of artefacts during dilution. The standard grid preparation method was used. As shown in Figure 5.3.1 the majority of particles appear small and round with a central pool of stain, resembling those seen in both the TatAd and TatE samples. Particles range in size from ~ 6 - 9.5 nm with the majority being ~ 8 nm in diameter.
Figure 5.3.1. Micrograph of TatAyCy-Strep undiluted gel filtration fraction 23 The micrograph was taken at ~57,000x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. TatAyCy forms a series of ringed particles varying in size from ~ 6.0 - 9.5 nm in diameter.
Chapter 5: Structural investigation of TatAyCy complexes
153 To improve particle separation the sample was diluted 1:5 using buffer + 0.02% DDM as for the TatAd sample (see chapter 4). As shown in Figure 5.3.2 the particle density is dramatically reduced, but the morphology of the particles is unaffected. Evidence of double-ringed structures was also observed; see Figure 5.3.3, despite the dilution, similar to those shown previously for the TatE sample (see chapter 5). This indicates that TatAyCy may be capable of modular multimerisation.
Figure 5.3.2. TatAyCy-Strep GF 23 1:5 dilution in GF buffer + 0.02% DDM 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
154 Figure 5.3.3. Evidence of multi-ringed structures within the TatAyCy sample A. Examples of double-ringed particles seen within the TatAyCy-strep sample are indicated by white arrows. A central pool of stain can be seen within each ring. These particles measure ~ 10 nm long and ~ 5 nm wide. B. Median filter applied to the same images as shown in A.
EM evaluation of gel filtration fractions 22 and 26 also displayed small ring shaped particles and did not reveal any discernible correlation between complex size and relative abundance of Tat subunits. Overall these data indicate that the Tat proteins are able to form stable, low order structures but the formation of TatAyCy complexes appears to be unstable under these conditions. The interaction between TatAy and TatCy is clearly evident from the biochemical data, but this interaction appears to be transient or unstable in nature.
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Chapter 5: Structural investigation of TatAyCy complexes
155 5.3.2. Higher order tetrameric assemblies of TatAyCy
When analysing gel filtration fraction 24 an interesting subset of particles were identified. As shown in Figure 5.3.4 small, ringed particles were again observed with no clear change in morphology from those of fraction 23. However, amongst these particles larger ringed structures with a striking tetrameric arrangement were also seen.
These particles were found infrequently (1 or 2 per micrograph at best) along with other large, but less defined structures, of a similar size; these potentially represent broken and flatten copies of the same particles. Examples of these larger structures are shown clearly in Figure 5.3.5. These well defined complexes consist of 4 copies of small ringed particles each measuring ~ 6 - 7 nm in diameter, the full tetramer is ~ 17 nm wide with a central stain pool of ~ 5.5 nm wide. This central ‘pore’ is larger than those seen for any of the TatAd or TatE particles.
Based on the purity of this sample (see Figure 5.2.1) and the similarity in size and shape of the component densities to the individual ringed particles, these tetramers appear to represent an unstable but highly ordered complex of Tat proteins. The relative contribution of subunits within the structure is unclear but they presumably consist of both TatAy and TatCy subunits.
These tetramers were not identified in any Tat samples where only the A (or A like) subunit is present and as such these data fit well with the observation that higher order oligomerisation of TatA normally requires the presence of the TatBC subunits (Leake et al., 2008). These particles are larger than other identified Tat complexes, are only found in the presence of all subunits required for translocation and are found in low abundance, which is indicative of an inherently unstable structure or a transient coalescence of subunits. Taken together these finding indicate that these tetramers may be representing a higher order assembly of the TatAyCy complex that could be of interest for future studies. However, until they can be isolated in greater numbers the biological significance of these assemblies cannot be verified.
Chapter 5: Structural investigation of TatAyCy complexes
156 Figure 5.3.4. TatAyCy-Strep GF 24 1:5 dilution in GF buffer + 0.02% DDM 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
157 Figure 5.3.5. Potential tetrameric complexes observed for TatAyCy-strep
A. Montage of large multimeric complexes of Tat protein. The top panels show clear tetramers of small ringed units, each ~ 5.5-7 nm wide, joined together by short extensions. A pool of stain is clearly visible within most of these small units. The central stain pool of the tetramer is ~ 5.5 nm wide. The lower panels show examples of distorted or broken complexes. B. The same particles shown in the top panel of A. are displayed with a median filter (middle panel) and as a binary outline image (lower panel) to clearly identify the modular structure.
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Chapter 5: Structural investigation of TatAyCy complexes
158 5.4. Purification of TatAyCy-his wild-type vs. P2A mutant
The results for the complex formation of the strep-tagged sample were interesting but unexpected. In order to further investigate these complexes by EM and to conduct Nanogold binding experiments a construct with a C-terminal Hexahistidine tag was used.
It has previously been shown that a range of single amino acid mutations in TatAy can abolish translocation of the substrate YwbN by TatAyCy in B. subtilis. (van der Ploeg et al., 2011). These mutations were shown to have variable effects on complex stability as seen by gel filtration but no further structural investigation was undertaken. Here the effects of substituting Pro-2 in the extracytoplasmic N-terminal region with Ala (P2A mutation) on complex formation and structure are shown. For these purifications larger samples of solubilised membranes (~ 11 ml) were provided, and a larger affinity column was used (10 ml Talon ). All other conditions and reagents used were the same as used for the strep-tagged sample.