Probing bulky ligand entry in engineered archaeal ferritins

Background
A set of engineered ferritin mutants from Archaeoglobus fulgidus (Af-Ft) and Pyrococcus
furiosus (Pf-Ft) bearing cysteine thiols in selected topological positions inside or outside the ferritin
shell have been obtained. The two apo-proteins were taken as model systems for ferritin internal
cavity accessibility in that Af-Ft is characterized by the presence of a 45 Å wide aperture on the
protein surface whereas Pf-Ft displays canonical (threefold) channels.
Methods
Thiol reactivity has been probed in kinetic experiments in order to assess the protein matrix
permeation properties towards the bulky thiol reactive DTNB (5,5'-dithiobis-2-nitrobenzoic acid)
molecule.
Results
Reaction of DTNB with thiols was observed in all ferritin mutants, including those bearing
free cysteine thiols inside the ferritin cavity. As expected, a ferritin mutant from Pf-Ft, in which the
cysteine thiol is on the outer surface displays the fastest binding kinetics. In turn, also the Pf-Ft mutant
in which the cysteine thiol is placed within the internal cavity, is still capable of full stoichiometric
DTNB binding albeit with an almost 200-fold slower rate. The behaviour of Af-Ft bearing a
cysteine thiol in a topologically equivalent position in the internal cavity was intermediate among
the two Pf-Ft mutants.
Conclusions and General Significance
The data thus obtained indicate clearly that the protein matrix in archaea ferritins does not
provide a significant barrier against bulky, negatively charged ligands such as DTNB, a finding of
relevance in view of the multiple biotechnological applications of these ferritins that envisage ligand
encapsulation within the internal cavity.