ESTABLECIOMIENTO DE 4 VARIEDADES DE MAIZ (Zea mays)

bound L7/L12

Residual dipolar couplings are NMR parameters that can be measured with high sensitivity and that provide high-resolution structural information, in terms of both the orientations of individual inter-nuclear vectors within the protein structure and the global orientational probability distribution of the protein under anisotropic conditions. In order to explore the possibility of applying an RDC approach to the study of NCs, the technique was first applied to the dynamic L7/L12 stalk region of the ribosome, with a view to developing the required methodology for the subsequent study of RNCs. The rationale behind this strategy was based on the similar motional properties that are expected for the two dynamic, ribosome-bound species.

As described in Chapter 2, the development of an RDC strategy to derive alignment tensors and local structural information for L7/L12 on the 70S ribosome was largely dictated by the challenges associated with the low maximum working concentration of the ribosome (10 µM). This consideration narrowed the choice for the types of RDC that could be measured, with the focus on intrinsically large couplings (N-H and Cα-Hα) and those that, while smaller, can be measured with high sensitivity (N-C’ and C’-Cα).

The alignment of the ribosome was successfully achieved using phage, and with the above considerations in mind, N-H RDC measurements were acquired using an HSQC/TROSY strategy. This approach was found to be optimal in this case because of the associated sensitivity and resolution gains compared to the IPAP-HSQC approach, which is more commonly applied to small, isolated proteins. The use of Cα-Hα couplings was

also explored because of their intrinsically large size. Modifications of established pulse sequences were implemented in an attempt to optimise the sensitivity and resolution for examining the ribosome-bound L7/L12.

To complement the N-H measurements made in phage, efforts to acquire RDCs in an alternative alignment medium were also made. Due to its large size and intrinsic lability, aligning the ribosome is not as straightforward as for simple proteins. The Otting medium, based on polyethylene glycol, was selected as a promising candidate for a second alignment medium, since it is reported to be inert towards most proteins, and should provide a near orthogonal alignment tensor to that produced in phage. However, the alignment was not as effective as with phage, as the ribosome appeared to interfere with the formation of the liquid crystal phase, although further attempts at lower concentrations could prove successful.

The N-H RDCs measured in phage using the HSQC/TROSY approach were able to yield detailed information on the ribosome-bound L7/L12. The measurement of the Cα-Hα RDCs was more challenging, with limited resolution in the13Cα-HSQC of ribosome-bound L7/L12 preventing extraction of splittings for many of the residues. The measurement of isotropic splittings for the H-C’/N-C’ couplings showed that it may be feasible to measure the N-C’ RDCs with sufficient precision and sensitivity to provide useful information. An attempt to measure the C’-Cα RDCs was unsuccessful, due to the low sensitivity associated with the ribosome-bound L7/L12. Indeed, the results of this study suggest that given the current sample conditions suitable for ribosomes, a future prospect for the measurement of ribosomal RDCs may lie in an alternative choice of organism. Thermophilic ribosomes (e.g. from Thermus thermophilus) have proved to be instrumental in crystallographic studies, presumably due to the inherent stability of the complexes. The enhanced thermostability may present the opportunity to either increase the concentration or, more likely, extend the lifetime of the 70S complex. As the L7/L12 stalk protein is highly conserved, presumably so too are its dynamic properties, and hence the development of RDCs in this context may offer alternative avenues of exploration.

L7/L12, allowing a comparison of its structural characteristics in isolation and when bound to the ribosome. A concentration-dependent oligomerisation of isolated L7/L12, postulated to be a dimer-tetramer interconversion, was observed. The oligomerisation event appeared to be mediated by the N-terminal domain of the L7/L12 protein and complicated RDC analysis due to the exchange between the different oligomeric states. For ribosome-bound L7/L12, the NTD is NMR-invisible and its structure could not be studied. The interpretation of the RDC data required careful consideration to take into account the intrinsic dynamics of L7/L12, so that only data from the rigid regions of the protein were considered.

The study of the CTD of isolated and ribosome-bound L7/L12 provided insights into both its structure and orientational preferences in the two states. The alignment tensors of the CTDs from the two gel-filtration fractions were very similar. This result is likely to arise from a combination of the weak alignment of the different NTDs and the fact that the alignment of the CTD will be dictated primarily from its own interaction with the phage, so that any influence from the NTD that may be transmitted through the linker region is so small as to be undetectable. The alignment tensors for the isolated and ribosome-bound CTDs were slightly different (although not to a highly significant degree), which may provide evidence for the orientation of the ribosome, and hence ribosome-bound NTD, affecting that of the L7/L12 CTD. Interestingly, however, only one set of RDCs was observed for the ribosome-bound L7/L12, despite the current understanding that the NMR signal arises from two CTDs, one on each of the two L7/L12 dimers. This suggests that there may be exchange of the dimers between the two ribosome-binding sites on the L10 protein or that the influence of the ribosome on the orientational preference of the extended CTDs is very small. Further studies using lanthanide-based alignment should yield more information on the flexibility of the linker region by revealing more clearly the relative orientational preferences of the N- and C-terminal domains.

The measurement of the N-H RDCs in phage also indicated that the structure of the L7/L12 CTD is very similar in the isolated and ribosome-bound states, and this similarity was confirmed by a limited structural refinement using the RDC data. Improvement of the

refinement protocol requires measurement of the N-H RDCs in another alignment medium. Attempts in this direction with the Otting medium were as yet only partly successful, but with indications that modifications could lead to improved alignment.