There is much contemporary interest in harnessing the capacity of protein macromolecules to self-assemble in order to construct smart materials, responsive surfaces or nanodevices. Currently the Lsm protein system, which spontaneously self-assembles into 75 Å ring structures, is being engaged as a potential model nanostructure with the ability to bind and sequester RNA. Our particular interest is to control the quaternary organisation of Lsm toroidal building blocks for ultimate fabrication into array and stacked tubule-like architectures.

Synthetic bioengineering techniques have been applied to generate simplified Lsm polyproteins fused via a Gly-rich linker sequences. These Lsm building modules readily organise into either single rings of tetramers or overlaid pairs of rings.

I outline the isolation of discrete Lsm polyproteins by size-exclusion chromatography (SEC) as well as their biophysical characterisation. Small-angle x-ray scattering (SAXS) as well as electron microscopy (EM) are being employed to investigate quaternary structure. These methods confirm discrete ring organisations are being formed by several distinct polyprotein groupings.

Investigation of solvent conditions has revealed that single rings of specific constructs are favoured by low salt. Their association into larger ring stacks has been determined to be under electrostatic control. We are proceeding to engineer mutant sequences to optimise higher-order robust rings, involving the inclusion of crosslinking-selective residues.