Further exploration and colonization of space demand tools for rapidly and portably monitoring hormones, metabolites, and other important biomarkers in astronauts. Although many of the techniques used terrestrially to measure these biochemicals are incompatible with spaceflight, plasmonic sensors deposited on paper could provide a suitable analytical platform if further improvements are made to the sensitivity of these nanomaterials. While multiple approaches have been demonstrated to improve the sensitivity of plasmonic sensors, even greater improvements could be attained by combining these separate approaches using a molecular scaffold capable of enduring elevated temperatures and assembling gold nanoparticles with nanoscale resolution. We report the development of such scaffolds using a largely unexplored strategy of covalently linking multiple individually folded proteins through site-specific ligations. Specifically, we employed split inteins to post-translationally ligate multiple copies of a rigid three-helix bundle protein into unimolecular protein nanostructures with triangular and square shapes. Coarse-grained simulations indicated that the length of the flexible peptide linkers connecting the 3HB “edges” of these structures determined the rigidity and uniformity of these protein shapes; we consequently optimized structural regularity by minimizing the length of these linkers. These covalently assembled nanostructures displayed high thermostability and resisted denaturation by chaotropes and organic solvents. We also conjugated gold nanoparticles to the vertices of these structures, demonstrating their potential as molecular scaffolds not only for constructing the advanced plasmonic sensors as proposed but also for applications in fields such as catalysis, signal processing, and drug delivery.

Construction of ultrastable protein nanoscaffolds for the assembly of spaceflight-compatible biosensors

ACS Fall 2020 Virtual Meeting

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