We have set up a tool to detect and monitor ferritin self assembly using FRET, which allows a better understanding on the pathway and kinetics of ferritin assembly and inter-subunit associations. We synthesized a batch of fluorescent ferritins, both H- and L-type, and studied ferritin self-assembly by different approaches. We found that when equimolecular amounts of donor and acceptor fluorophores were coassembled, FRET efficiency raised from values of ̴0.3 on the disassembled state to ̴0.7 after assembly, confirming an intersubunit fluorescence energy transfer (FRET) on the assembled state in accordance with inter-subunit distances. Studies carried out on the kinetics of assembly showed that both the H-chain homopolymers and H/L heteropolymers have similar patterns and complete the assembly process in about one hour. On the other hand, preliminary results suggest a different pattern for subunit distribution that is dependent on the homopolymeric or the heteropolymeric nature of reassembled ferritin.

1. Conclusions
We have set up a tool to detect and monitor ferritin self-
assembly using FRET, which allows a better understanding on
the pathway and kinetics of ferritin assembly and inter-subunit
associations. We synthesized a batch of fluorescent ferritins,
both H- and L-type, and studied ferritin self-assembly by
different approaches. We found that when equimolecular
amounts of donor and acceptor fluorophores were co-
assembled, FRET efficiency raised from values of ̴0.3 on the
disassembled state to ̴0.7 after assembly, confirming an inter-
subunit fluorescence energy transfer (FRET) on the assembled
state in accordance with inter-subunit distances. Studies carried
out on the kinetics of assembly showed that both the H-chain
homopolymers and H/L heteropolymers have similar patterns
and complete the assembly process in about one hour. On the
other hand, preliminary results suggest a different pattern for
subunit distribution that is dependent on the homopolymeric or
the heteropolymeric nature of reassembled ferritin.
Aims
The aim of this work is to study the mechanisms and kinetics of ferritin self-assembly
and homo- and heteropolymer formation using a dynamic tool such as FRET, that
allows the monitoring of protein dynamics and thus may give light on the
development of more versatile heteropolymeric ferritins with different functionalities
for biomedical applications.
Materials and Methods
• Synthesis and characterization of covalently bound fluorescent AlexaFluor-Ferritin
derivatives. UV/Vis and fluorescence spectroscopy showed that the number of
fluorophores covalently bound to exposed Lys or Cys accounted to about one per
ferritin subunit.
• The fluorescent ferritins were then denatured and reassembled in different proportions,
excited at the wavelength of the donor fluorophore, and the efficiency of FRET
expressed as Ratio A [4].
• Kinetic and structural studies on ferritin self-assembly were carried out using
fluorescence spectroscopy and PAGE.
References
Samples
Ferritin labeling reactions were done by targeting exposed cysteine
(FTH) and lysine (FTH and FTL) residues on the outer surface of
ferritins. Targeted coupling was achieved using AlexaFluor488-
maleimide (Cys) and AlexaFluor555-succinimidyl esther (Lys)
derivatives, which in addition are suitable to undergo FRET because
of their overlapping spectra:
Kinetics of ferritin self-assembly
Gel Filtration elution profiles:
FTH and FTL homopolymers were incubated for 3h at room
temperature with an excess of the corresponding AlexaFluor
derivative. Non bound fluorophores were then removed with
extensive dialysis and separated on a Superose 6 column. Elution
profiles of the samples show that both the protein and the
fluorophore elute together. Non-denaturing gel electrophoresis
scanned with UV light before being stained with Coomassie
confirmed the covalent binding of the AlexaFluors to the protein
surface.
Purification
In a general experiment, the fluorescent ferritin homopolymers are
disassembled (1) by denaturation with Guanidine HCl 6M pH 3.5
and then, mixed in a 1:1 proportion of DONOR:ACCEPTOR
ferritin, i.e. FTH488:FTH555 (homopolymer) or FTH488:FTL555
(heteropolymer). The unfolded ferritin mixture is then reassembled
(2) by 10-fold dilution with Tris 0.1 M pH 8 or left disassembled by
10-fold dilution with Guanidine HCl 6M pH 8.
When AF-labeled ferritins are co-assembled, the electrophoretic motility confirms
the co-assembly of the different subunits as revealed by UV scanning and
Coomassie staining. The FRET efficiency obtained is dependent on the
DONOR:ACCEPTOR ratio showing higher efficiencies at lower acceptor
proportions.
Monitoring self-assembly by FRET
Equimolar amounts of unfolded FTH488 and FTH555, or FTH488 and FTL555,
were mixed and then re-assembled to yield the corresponding fluorescent
homopolymers (H/H) or heteropolymers (H/L). The results show that when donor
and acceptor fluorophores are co-assembled, FRET efficiency, expressed as (ratio)A
raises to ̴ 0.7, confirming the co-assembly of the subunits in the same protein shell.
In addition, both the unfolded ferritins and a mixture of not co-assembled
homopolymers yielded remarkably decreased FRET efficiencies, confirming thus
the suitability of this tool for monitoring ferritin self-assembly.
Monitoring of ferritin self-assembly
by Fluorescence Resonance Energy Transfer (FRET)
Fernando Carmona, Maura Poli, Magdalena Gryzik, Andrea Denardo, Paolo Arosio
Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy
fernando.carmonarodriguez@unibs.it
Homopolymer
Kinetics of homopolymeric (H/H) and heteropolymeric (H/L) ferritin self-assembly was studied using
FRET. Equimolar mixtures of FTH488:FTH555 and FTH488:FTL555 were refolded and the efficiency of
FRET monitored on time until no changes in Ratio A were observed. The results reveal the assembly
process on time for the first time, and show thatthat both kinetics of the assembly for H-chain
homopolymers and H/L heteropolymers have similar patterns and complete the assembly process in about
one hour, in agreement with PAGE results.
Subunit-type dependent localization on ferritin homopolymers and heteropolymers
Hypothesis
Whether there is a preferential association of subunit heterodimers (H/L) over
homodimers (H/H) in the process of self-assembly is not yet clarified.
Considering this, and being the FRET efficiency strongly dependent on the
distance between donor and acceptor, we produced a batch non-labeled H and L
ferritins re-assembled with increasing amounts of HH (Donor-Acceptor) FRET-
couples. We hypothesize that, given the H-chain nature of the HH (Donor-
Acceptor) FRET-couple, the reassembly on a H-homopolymer would lead to a
random distribution of the fluorescent subunits, as they are “diluted” on a full
H-type ambient, in contrast with the heteropolymer, where L-type subunits are
present.
Introduction
Ferritins are highly conserved proteins designed to store iron and attenuate hydroxyl radical generation from the
Fenton reaction [1]. Their structure is an almost spherical nanocage made of 24 subunits delimiting a large cavity.
The high stability and strong self-assembly properties have made ferritins the most used proteins for nano-
technological applications [2]. However, the mechanism and kinetics of ferritin self-assembly and homo- and
heteropolymers formation remains unclarified. To study ferritin self-assembly we used Fluorescence Resonance
Energy Transfer (FRET) [3,4], by binding multiple donor or acceptor AlexaFluor fluorophores on the outer surface
of human H and L ferritin chains and the resulting fluorescent ferritins were then denatured and reassembled in
different proportions. The results show that when co-assembled, FRET efficiency raises to ̴ 0.7, confirming an inter-
subunit fluorescence energy transfer (FRET) in accordance with inter-subunit distances. This allowed us to carry on
studies on the kinetics of self-assembly using FRET, finding that both kinetics of the assembly for H/H
homopolymers or H/L heteropolymers have similar patterns and complete the assembly process in about one hour,
in agreement with PAGE results. Furthermore, being the FRET highly dependent on the distance between donor
and acceptor, we question whether there is a preferential association of subunit heterodimers (H/L) over
homodimers (H/H) in the process of self-assembly. Preliminary results suggest a different pattern for subunit
distribution that is dependent on the homopolymeric or the heteropolymeric nature of the reassembled ferritin.
Further experiments are currently in progress to verify the nature of subunits association on different ferritin shells.
Ferritin-type dependent distribution of H-
chains
[1] Arosio, P. et al. Biochem. J., 2015.
[2] Jutz, G. et al. Chem. Rev., 2015.
[3] Carmona, F. et al. JBIC, 2014.
[4] Sternberg, S. et al. Nature, 2015.
Reaction Scheme
Heteropolymer
Ferritin is made up of 24 subunits
that co-assemble to form a highly
symmetric dodecahedron 12 nm in
diameter. Subunit-subunit dimers
create 2-fold axes. Eight
hydrophilic channels pierce the 3-
fold axes allowing Fe(II) difussion
to the cavity and 6 hydrophobic
channels in the 4-fold axes
complete the structure of ferritin.
Int.Emiss.
Is that FRET?? Ratio A
FRET Efficiency is commonly expressed as Ratio A [4]. The basis
of Ratio A is to obtain the ratio between the emission obtained
because of FRET by directly excitating to the DONOR, divided
into the full emission intensity of the system directly excitating at
the ACCEPTOR fluorophore. Thus, Ratio A ranges from 0 to 1.
The titration of non-labebeled FTH and FTL with increasing amounts of FRET couples i.e.: 2
H488 subunits + 2 H555 subunits reveal a different pattern of localization of the FRET subunits
on the structure of the final assembled ferritin depending on its homopolymeric (H/H) or
heteropolymeric (H/L) nature. When assembling an homopolymer, FRET couple subunits (also
H-type) arrange randomly on the structure yielding thus a linear pattern on the Ratio A, that is
directly dependent on the total amount of FRET subunits present in the final structure. However,
when the FRET subunits (H-type) are assembled on a L-type structure (heteropolymer), a
sigmoidal pattern appears, suggesting that the first 8 H- subunits localize at distant positions (low
Ratio A) and then, increasing H-subunits crowd the structure resulting in a jump on the Ratio A.
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