title: Probing bulky ligand entry in engineered archaeal ferritins.
abstract: 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.
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Calisti
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Benni
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Matilde
authors.initials: M
authors.lastname: Cardoso Trabuco
authors.affiliation: Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, V.le Regina Elena 291, Rome I-00185, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Baiocco
authors.affiliation: Istituto dei Sistemi Complessi del Consiglio Nazionale delle Ricerche (ISC-CNR) Sede Sapienza and Dipartimento di Fisica, Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ruzicka
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy; Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche (IBPM-CNR) Sede Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Boffi
authors.affiliation: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche (IBPM-CNR) Sede Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Falvo
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Malatesta
authors.affiliation: Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy. Electronic address: alessandra.bonamore@uniroma1.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bonamore
mesh_terms: D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism*; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D008024:Ligands; D013438:Sulfhydryl Compounds; D005293:Ferritins; D004228:Dithionitrobenzoic Acid; D003545:Cysteine
keywords: Binding kinetics; Ferritin; Nano-scaffold; Self-assembly