Engineering peroxiredoxin 3 to facilitate control over self-assembly.

Oligomeric proteins are abundant in nature and are useful for a range of nanotechnological applications; however, a key requirement in using these proteins is controlling when and how they form oligomeric assemblies. Often, protein oligomerisation is triggered by various cellular signals, allowing for controllable oligomerisation. An example of this is human peroxiredoxin 3 (Prx), a stable protein that natively forms dimers, dodecameric rings, stacks, and tubes in response to a range of environmental stimuli. Although we know the key environmental stimuli for switching between different oligomeric states of Prx, we still have limited molecular knowledge and control over the formation and size of the protein's stacks and tubes. Here, we have generated a range of Prx mutants with either a decreased or knocked out ability to stack, and used both imaging and solution studies to show that Prx stacks through electrostatic interactions that are stabilised by a hydrogen bonding network. Furthermore, we show that altering the length of the polyhistidine tag will alter the length of the Prx stacks, with longer polyhistidine tags giving longer stacks. Finally, we have analysed the effect a variety of heavy metals have on the oligomeric state of Prx, wherein small transition metals like nickel enhances Prx stacking, while larger positively charged metals like tungstate ions can prevent Prx stacking. This work provides further structural characterisation of Prx, to enhance its use as a platform from which to build protein nanostructures for a variety of applications.

Formation of supramolecular protein structures on gold surfaces.

Recent research has highlighted the exciting possibilities enabled by the use of protein structures as nanocomponents to form functional nanodevices. To this end, control over protein-protein and protein-surface interactions is essential. In this study, the authors probe the interaction of human peroxiredoxin 3 with gold surfaces, a protein that has been previously identified as having potential use in nanotechnology. Analytical ultracentrifugation and transmission electron microscopy revealed the pH mediated assembly of protein toroids into tubular structures across a small pH range. Quartz crystal microbalance with dissipation measurements showed differences in absorbed protein mass when pH is switched from pH 8.0 to 7.2, in line with the formation of supramolecular structures observed in solution studies. Scanning tunneling microscopy under ambient conditions showed that these protein tubes form on surfaces in a concentration dependent manner, with a tendency for protein adsorption and supramolecular assembly at the edges of Au(111) terraces. Finally, self-assembled monolayer modification of Au surfaces was explored as a means to control the adsorption and orientation of pH triggered protein structures.

Proteins as supramolecular building blocks: Nterm-Lsr2 as a new protein tecton.

Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. We have explored self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the N-terminal domain of Lsr2, Nterm-Lsr2. A key feature of this protein is that it undergoes self-assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm-Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm-Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed.