Probing bulky ligand entry in engineered archaeal ferritins

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 2; pp. 450 - 456
• Main Authors: Calisti, Lorenzo, Benni, Irene, Cardoso Trabuco, Matilde, Baiocco, Paola, Ruzicka, Barbara, Boffi, Alberto, Falvo, Elisabetta, Malatesta, Francesco, Bonamore, Alessandra
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2017
• Subjects: Archaea - genetics; Archaea - metabolism; Archaeoglobus; Archaeoglobus fulgidus - genetics; Archaeoglobus fulgidus - metabolism; Binding kinetics; cysteine; Cysteine - metabolism; Dithionitrobenzoic Acid - metabolism; encapsulation; Ferritin; Ferritins - genetics; Ferritins - metabolism; Kinetics; Ligands; mutants; Mutation - genetics; Nano-scaffold; Pyrococcus furiosus; Pyrococcus furiosus - genetics; Pyrococcus furiosus - metabolism; Self-assembly; Sulfhydryl Compounds - metabolism; thiols; topology; Self-assembly; Binding kinetics; Nano-scaffold; Ferritin
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.10.007

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 45Å wide aperture on the protein surface whereas Pf-Ft displays canonical (threefold) channels. 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. 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. 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. •A set of engineered archael ferritin mutants have been obtained.•The mutants bear cysteine thiols in selected topological positions.•Thiol reactivity has been probed using the bulky thiol reactive DTNB.•Archaeal ferritins can bind DTNB outside but also inside the cavity.•Protein matrix is not a significant barrier against bulky, negatively charged ligands.