Investigation of lsm proteins as scaffolds in bionanotechnology

Self-assembling materials have gained attention in the field of nanotechnology due to their potential to be used as building blocks for fabricating complex nanoscale devices. The biological world is abundant with examples of functional self-assembling biomolecules. Proteins are one such example, found in a variety of geometries and shapes. This research is focussed on the use of ring-shaped self-assembling proteins, called Lsm proteins, as componentary for applications in bionanotechnology. Lsm proteins were used because of their spontaneous association into stable rings, tolerance to mutations, and affinity to RNA. This thesis primarily focussed on the thermophilic Lsmα (from Methanobacterium. thermoautotrophicum) that assembles as heptameric rings. The oligomeric state of the heptameric protein, and hence the diameter of its central cavity, was manipulated by judiciously altering appropriate residues at the subunit interface. Lsmα presented a complex set of interactions at the interface. Out of the mutations introduced, R65P yielded a protein for which SEC and SAXS data were consistent with a hexameric state. Moreover, key residues, L70 and I71, were identified that contribute to the stability of the toroid structure. Covalent linking of rings provided nanotubular structures. To achieve this, the surface of the Lsmα ring scaffold was modified with Cys residues. This approach led to the formation of novel Lsmα nanotubes approximately 20 nm in length. Importantly, the assembly could be controlled by changing the redox conditions. As an alternative method to manipulate the supramolecular assembly, His6-tags were attached at the termini of the Lsmα sequence. The higher-order organisation of the constructs was influenced by the position of the His6-tag. The N-terminally attached His6-tag version of Lsmα showed a metal-dependent assembly into cage-like structures, approximately 9 nm across. This organisation was highly stable, reproducible, and reversible in nature. The results presented in this thesis aid the understanding of generating complex nanostructures via in vitro self-assembly. The Lsmα rings were assembled into higher-order architectures at the quaternary level by employing protein engineering strategies. Future work is necessary to functionalise these supramolecular structures; however, this study confirms the potential role of Lsmα proteins as a molecular building block in bionanotechnology