Lorenzo Calisti1, Irene Benni1, Matilde Cardoso Trabuco1, Paola Baiocco2, Barbara Ruzicka3, Alberto Boffi4, Elisabetta Falvo5, Francesco Malatesta1, Alessandra Bonamore6. 1. Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy. 2. Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, V.le Regina Elena 291, Rome I-00185, Italy. 3. 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. 4. 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. 5. 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. 6. 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.
Abstract
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 45Å 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.
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 45Å 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.
Authors: Lorenzo Calisti; Matilde Cardoso Trabuco; Alberto Boffi; Claudia Testi; Linda Celeste Montemiglio; Amédée des Georges; Irene Benni; Andrea Ilari; Bartłomiej Taciak; Maciej Białasek; Tomasz Rygiel; Magdalena Król; Paola Baiocco; Alessandra Bonamore Journal: PLoS One Date: 2018-08-13 Impact factor: 3.240