Crystallizing RNA has been an imperative facet and a challenging task in the world of RNA research. Assistive methods such as Chaperone Assisted RNA Crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by increasing the crystal contacts and providing initial phasing information. Using this technology the crystal structure of [delta]C209 P4-P6 RNA (an independent folding domain of the self-splicing Tetrahymena group I intron) complexed to Fab2 (high affinity binding Fab) has been resolved to 1.95 Å (1). Although the complexed class I ligase ribozyme has also been crystallized using CARC (2), in practice, it has been found that the crystallization of, large RNA-Fab complex remains a confrontation. The possible reason for this difficulty is that Fabs have not been optimized for crystallization when complexed with RNA. Here we have used the Surface Entropy Reduction technique (SER) for the optimization process. Candidate residues for mutations were identified based on combining results from visual inspection of [delta]C209 P4-P6/Fab2 crystal structure complex using pyMOL software and a web-based SER software. The protruding lysine and glutamate residues were mutated to a set of alanine (Super Mutant Alanine SMA) and serine (Super Mutant Serine SMS) mutant clones. Filter binding assay studies confirmed that the mutant clones bind to [delta]C209 P4-P6 with similar binding affinities as that of the parent Fab2. Large scale expression of the mutants, parent clone and [delta]C209 P4-P6 RNA were optimised. Crystal trays for [delta]C209 P4-P6 complexed with Fab2, Fab2SMA and Fab2SMS were set-up side-by-side using Hampton crystal screen kits and ~600 conditions including temperature as a variable condition were screened. Crystal screening shows significantly higher crystal-forming ratios for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can be potentially applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of the Fab-RNA complex.

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Catechol 1,2-Dioxygenase from Rhodococcus opacus is a type of intradiol dioxygenase enzyme that catalyzes the conversion of catechol to cis, cis muconic acid. This enzymatic conversion has the potential to be useful in a number of different applications such as treating wastewater contaminated with aromatic compounds to creating a greener method to produce cis, cis muconic acid which can be used to make a number of industrially important base chemicals. However, for enzymes to be used in industrial conditions, they must be highly stable. The experimental chapters in this thesis explore whether this enzyme can be stabilized to meet industrial requirements while minimizing any loss in catalytic activity. Through the studies described in Chapter 2, a mutant enzyme was generated through disulfide bond engineering with significantly improved thermostability. However overall catalytic activity was reduced. Toward addressing this loss of catalytic activity, in Chapter 3, attempts were made to implement state-of-the-art genetic code expansion strategies to increase catalytic activity of the enzymes. However, these attempts were unsuccessful. Finally, Chapter 4 describes how future stability engineering could be optimized using design pipelines similar to the one developed in this study. Additionally, it describes possible additional optimizations toward making the application of these enzymes cost effective in the near future.

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