Asymmetric bifunctional protein nanoparticles through redesign of self-assembly

Abstract

Engineering oligomeric protein self-assembly is an attractive approach to fabricatenanostructures with well-defined geometries, stoichiometry and functions. The homodecamerBrucella Lumazine Synthase (BLS) is a highly stable and immunogenic protein nanoparticle (PNP).Here, we engineered the BLS protein scaffold to display two functions in spatially opposite regions ofits structure yielding a Janus-like nanoparticle. An in silico analysis of the BLS head-to-head dimer ofhomopentamers shows major inter-pentameric interactions located in the equatorial interface. Basedon this analysis, two BLS protomer variants were designed to interrupt pentamer self-dimerizationand promote heteropentameric dimers. This strategy enabled us to generate a decameric particle withtwo distinct sides formed by two independent pentamers. The versatility of this new self-assemblynanofabrication strategy is illustrated with two example applications. First, a bifunctional BLSbearing Alexa Fluor 488 fluorophores on one side and sialic acid binding domains on the other sidewas used for labelling murine and human cells and analyzed by flow cytometry and confocalmicroscopy. Second, multichromophoric FRET nanoparticles were fabricated and characterized at thesingle molecule level, showing discrete energy transfer events. The engineered BLS variantsconstitute a general platform for displaying two functions in a controlled manner within the same PNPwith potential applications in various areas such as biomedicine, biotechnology and nanotechnology.