The structural characterisation of macromolecules via X-ray crystallography requires the generation of diffracting protein crystals. However not all proteins are amenable to crystallisation, thereby presenting a rate-limiting step in the technique. High-resolution structural information affords a comprehensive understanding of protein function at atomic-resolution. Such insight can directly contribute to the generation of therapeutics or contribute to the understanding of a biochemical pathway. The advent of rapid genome sequencing has identified a large number of desirable structural target proteins, generating a bottleneck at crystallogenesis. While a multitude of additives aimed at improving crystallogenesis have been reported, no generic additives have yet been identified. Thus the research presented in this thesis aimed to develop generic additives to improve crystallogenesis and describe work on the structural characterisation of a particularly troublesome protein family. Of the 30,000 known pentatricopeptide repeat (PPR) proteins, no structural data was available at the outset of this project due to difficulties in crystallising these proteins via traditional methods. PPR proteins are essential for organelle biogenesis, RNA editing and posttranscriptional maturation in plants. With genetic mutations resulting in cytoplasmic male sterility, seed development and other phenotypic impairments. Recent developments in understanding the modular nature of sequence-specific RNA binding by PPR proteins have raised the possibility of de novo design of proteins with specific targets, for use in agricultural and medical biotechnology.
Study I and II focus on the generation of heterogeneous additives to improveprotein crystal generation. Using high-throughput crystallisation, screening, visualisation and X-ray diffraction we determined the efficacy of colloidal graphenes and nacre micro-tablets as heterogeneous additives and present their application as generic additives for improving protein crystal yield - successfully for graphene and unsuccessfully for nacre - using an objective, statistically-sound procedure and analysis to establish additive efficacy.
Study III and IV investigate the use of cavitands, small molecules capable of binding moieties in a central hydrophobic cavity, to improve protein stability and use host-guest interactions to propagate lattice formation during crystallisation trials. The studies included in this thesis describe necessary characterisation of the solution structure of cavitands as derived via comprehensive small angle X-ray scattering, ab initio modelling, crystal structures and co-crystallisation trials with cavitands and proteins. Such characterisation is required in order to exploit the vast potential of cavitands to augment structural biology in the future.
In study V native ligands were trialled to improve the conformational stability of a target protein destined for crystallisation trials. These studies include biophysical characterisation including circular dichroism and small angle X-ray scattering, ab initio modelling and informed global rigid body modelling. Combining these techniques we propose the global structure of a target PPR protein in complex with RNA. Utilising techniques developed during this thesis, conformational stability of the target protein was improved leading to the first monomeric structure of a PPR protein upon binding RNA. These studies highlight the importance of conformational stability in structural characterisation of proteins.
Study VI describes the de novo design of a synthetic PPR protein engineered to be more amenable to crystallisation, using sequence optimisation, biophysical methods, high-throughput crystallisation attempts and X-ray crystallography. This study describes the atomic-resolution crystal structure of the first synthetic PPR protein, offering potential as a scaffold for designing RNA binding PPR proteins and highlighting the importance of sequence optimisation in the crystallogenesis of troublesome proteins.
Overall, these studies reveal novel methodologies for improved crystallogenesis and structural stabilisation of target proteins. We used pre-existing and novel methodologies developed here, in a target-orientated crystallisation project to obtain highly sought-after comprehensive structural characterisation of members of the PPR protein family.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - May 2014|