title: Design of a Redox-Sensitive Supramolecular Protein Assembly System Operating in Live Cells.
abstract: A fusion construct between Citrine (a YFP variant) and human ferritin (H-chain) was recently shown to form supramolecular assemblies of micrometer size when expressed in mammalian cells. The assembly process is driven by weak hydrophobic interactions leading to dimerization of YFP. Protein assembly could be suppressed at the gene level by mutation in the primary sequence of the construct. In this work, we describe the engineering of a self-assembly interface sensitive to redox state in the cell. Key hydrophobic residues of YFP were mutated systematically to cysteines. Supramolecular assembly of the Citrine-ferritin construct was in some cases preserved by formation of disulfide bonds in place of hydrophobic interactions. In others cases, assembly was abolished, resulting in a diffuse distribution of the expressed protein. A specific variant that remained diffuse under normally reducing intracellular conditions was found to self-assemble rapidly upon exposure to a thiol-specific oxidizing reagent.
authors: Department of Materials and Interfaces, Weizmann Institute of Science , Rehovot 76100, Israel.
authors: Giuliano
authors: G
authors: Bellapadrona
authors: Department of Materials and Interfaces, Weizmann Institute of Science , Rehovot 76100, Israel.
authors: Michael
authors: M
authors: Elbaum
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D003593:Cytoplasm; D005293:Ferritins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C434479:citrine protein, bacteria; D005293:Ferritins
keywords: Supramolecular protein assembly; fluorescence microscopy; oligomerization; oxidation; protein−protein interaction