1. A FRET-based approach to characterize human ferritin self-assembly is described. 2. Self-assembly kinetics of H/L heteropolymers are faster than for H/H homopolymers. 3. Heterodimeric (H/L) association is preferred during H/L heteropolymers formation. 4. H-chains arrange preferentially at distant sites on H/L heteropolymeric shells.

1. STUDY OF FERRITIN SELF-ASSEMBLY AND
HETEROPOLYMER FORMATION USING FRET
Fernando Carmona, Maura Poli, Andrea Denardo, Michela Bertuzzi, Alessandra Gianoncelli, Fabrizio Gangemi, Paolo Arosio
Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy
fernando.carmonarodriguez@unibs.it
HIGHLIGHTS:
1. A FRET-based approach to characterize human ferritin self-assembly is described.
2. Self-assembly kinetics of H/L heteropolymers are faster than for H/H homopolymers.
3. Heterodimeric (H/L) association is preferred during H/L heteropolymers formation.
4. H-chains arrange preferentially at distant sites on H/L heteropolymeric shells.
+
H H
H/H +
H L
H/L
: Not labelled subunit
(H or L)
: Donor labelled-subunit (H) : Acceptor labelled-subunit
(H or L)
H/H HOMOPOLYMERS H/L HETEROPOLYMERS
2. KINETICS OF SELF-ASSEMBLY
1.
3&4. DISTRIBUTION OF H-CHAINS ON THE H/L HETEROPOLYMERIC SHELL
H/L
L
+
H H
+ n( )H/H
H
+
H H
+ n( )
+
H H
Number of labelled H-chains ( )
-Both kinetics of assembly are finished in about 1
hour.
-Assembly of H/L heteropolymers is slightly faster
than the H/H homopolymers.
-This, heterodimeric (H/L) association is dominant
than the homodimeric (H/H) one.
We used donor-acceptor H-chain couples (H-488 and H-555) as sensors of H-subunit proximity. The addition of controlled
amounts of donor-acceptor H-H couples can compare the effect of H- and L-chains in the assembly of the H-chains.
CONTROL: H-chains coassembled
with H-chains.
The intervening unlabeled H- chains
keep the donor and acceptor probes
far apart the FRET signal increases
linearly as the fluorescent H-chains
crowd the structure.
Since the energy transfer (ratio)A is an
index of subunit proximity, this result
indicates a random distribution of
the fluorescent H-chains not affected
by the intervening unlabeled H-
chains.
H-chains coassembled with L-chains.
This shows that the H-chain distribution in the
presence of L-chains is not random. H-chains
arrange distantly until a number of 8, and then start
to colocalize.
Negligible changes in FRET efficiency after the
addition 4, 6 and 8 H-chains, indicate that in the
presence of L-chains, the H-chains arrange at
distant positions on the shell until a number of 8.
This means that the L-chains displace the H- chains
during the formation of the subunit dimers,
indicating a preferred heterodimeric (H/L) over
homodimeric (H/H) association during H/L
heteropolymer formation.
+Fe(II)
+
H H
Number of labelled H-chains ( )
Iron uptake activity as a function of
Nº of H-chains
+
H H
Number of labelled H-chains ( )
Distant Co-localized
Max. activity
The FRET patterns of the heteropolymers indicated that the maximum
interference of the L-chains on the H-chains occurred in the 8H/16L hybrid,
where the H-chains are placed at distant positions.
Interestingly the iron uptake activity shows that these shells have the highest
capacity of iron incorporation and that a further increase in H- chains does not
increase the rate of iron incorporation.
In conclusion, the preferred formation of the subunit heterodimers facilitates
the activity of iron incorporation by putting the two major functions of iron
oxidation (H-chain) and of iron nucleation (L-chain) in close proximity.
Altogether the data indicate that the distribution of the ferroxidase activity,
i.e. the H-chains, in the ferritin shells has an impact on their functionality.
Iron uptake activity as a function of Nº of H-
chains.
We subjected the H/L heteropolymers described
above to iron uptake. The graph at the left shows
the Fe(III) core formation measured at 310 nm by
UV/vis.
Bibliography:
Carmona, F., Poli, M., Bertuzzi, M., Gianoncelli, A., Gangemi, F., & Arosio, P.
(2017), Biochimica et Biophysica Acta (BBA)-General Subjects, 1861(3), 522-532.