Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology
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AuthorCarmona Rodríguez-Acosta, Fernando; Poli, Maura; Bertuzzi, Michela; Gianoncelli, Alessandra; Gangemi, Fabrizio; Arosio, Paolo
FerritinSelf-assemblyHeteropolymersFluorescence energy transfer (FRET)
Carmona Rodríguez-Acosta, F.; et al. Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology. Biochimica et Biophysica Acta (BBA)-General Subjects 1861(3): 522-532 (2017). [http://hdl.handle.net/10481/47234]
SponsorshipThis work was partially supported by MIUR grant PRIN10-11 to PA, and by Telethon grant GGP15064 to PA. FC was recipient of a Post-Doc Fellowship from University of Brescia, and was partially supported by CIB (Consorzio Italiano di Biotecnologie).
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 12 nm 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.