title: Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology.
abstract: The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from <0.3 of the disassembled to >0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize.
authors.affiliation: Molecular Biology Laboratory, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.
authors.forename: Fernando
authors.initials: F
authors.lastname: Carmona
authors.affiliation: Molecular Biology Laboratory, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.
authors.forename: Maura
authors.initials: M
authors.lastname: Poli
authors.affiliation: Proteomics Platform, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Bertuzzi
authors.affiliation: Proteomics Platform, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Gianoncelli
authors.affiliation: Laboratory of Physics, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Gangemi
authors.affiliation: Molecular Biology Laboratory, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy. Electronic address: paolo.arosio@unibs.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Arosio
mesh_terms: D001052:Apoferritins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D011108:Polymers / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011108:Polymers; D001052:Apoferritins; D007501:Iron
keywords: Ferritin; Fluorescence energy transfer (FRET); Heteropolymers; Homopolymers; Self-assembly