Asymmetric bifunctional protein nanoparticles through redesign of self-assembly

SOSA, SANTIAGO; ROSSI, ANDRÉS H.; SZALAI, ALAN M.; KLINKE, SEBASTIÁN; RINALDI, JIMENA; FARIAS, ANA; BERGUER, PAULA M.; NADRA, ALEJANDRO D.; STEFANI, FERNANDO D.; GOLDBAUM, FERNANDO A.; BONOMI, HERNÁN R.

Nanoscale Advances

Royal Society of Chemistry

Año: 2019 vol. 1 p. 1833 - 1846

Engineering oligomeric protein self-assembly is an attractive approach to fabricate nanostructures withwell-defined geometries, stoichiometry and functions. The homodecamerBrucellaLumazine Synthase(BLS) is a highly stable and immunogenic protein nanoparticle (PNP). Here, we engineered the BLSprotein scaffold to display two functions in spatially opposite regions of its structure yielding a Janus-likenanoparticle. Anin silicoanalysis of the BLS head-to-head dimer of homopentamers shows major inter-pentameric interactions located in the equatorial interface. Based on this analysis, two BLS protomervariants were designed to interrupt pentamer self-dimerization and promote heteropentameric dimers.This strategy enabled us to generate a decameric particle with two distinct sides formed by twoindependent pentamers. The versatility of this new self-assembly nanofabrication strategy is illustratedwith two example applications. First, a bifunctional BLS bearing Alexa Fluor 488fluorophores on one sideand sialic acid binding domains on the other side was used for labelling murine and human cells andanalyzed byflow cytometry and confocal microscopy. Second, multichromophoric FRET nanoparticles were fabricated and characterized at the single molecule level, showing discrete energy transfer events.The engineered BLS variants constitute a general platform for displaying two functions in a controlledmanner within the same PNP with potential applications in various areas such as biomedicine,biotechnology and nanotechnology