PROTEIN SCAFFOLD LIBRARY FOR NANOTECHNOLOGICAL APPLICATIONS BASED ON BLS PROTEIN ENGINEERING

SOSA, SANTIAGO; SZALAI, ALAN M.; GOLDBAUM, FERNANDO A.; ROSSI, ANDRÉS HUGO; BONOMI, HERNÁN R.; VIOLI I; STEFANI, FERNANDO D.

La Plata, Buenos Aires.

Congreso; Reunion Anual SAB 2018; 2018

SAB

Self-assembly of biological macromolecules, such as proteins, lipids and DNA, is a key aspect of life. Protein nanoparticles (PNPs) are a special subset of supramolecular protein assemblies. The PNP hallmark is a symmetric, repetitive structure, that can have enhanced stability (e.g. chemical, mechanical or thermal) and can be highly immunogenic. Engineering oligomeric protein self-assembly is emerging as an interesting way to fabricate nanostructures with well-defined geometries, stoichiometry and functions.BLS (Brucella Lumazine Synthase) is a highly stable and immunogenic homodecamer that can be regarded as a dimer of head-to-head oriented homopentamers. Therefore, we decided to engineer it in order to develop a versatile scaffold. We have designed two pentameric mutants that can bind to each other to form an heterodecamer. Our results show that this heterodecameric structure can be used as an assymetric particle. As a proof of concept a bifunctional BLS particle bearing Alexa Fluor 488 fluorophore molecules on one side and sialic acid binding domains on the other was used for labelling murine and human cells (flow cytometry and confocal microscopy). We have also introduced reactive cysteines in different positions of the wild type BLS and its pentameric forms in order to evaluate different applications. We have some promising results (absorbance spectroscopy, DLS and SEM) that show that BLS can be used as a scaffold of Gold Nanoparticles oligomers. On the other side, we have shown that this structure can be used for multichromoforic FRET studies (smFRET). Finally, bioinformatics studies suggest that BLS can be used as a protein cage of small molecules. In silico analysis show that this protein has a cavity (9,5 Å radius) that can be engineered to bind fluorophores or drugs.In conclusion, we have developed a library of rational designed mutants of BLS that can be used in different fields, such as biotechnology and nanotechnology.