Probing bulky ligand entry in engineered archaeal ferritins.
Biochim Biophys Acta Gen Subj
; 1861(2): 450-456, 2017 Feb.
Article
em En
| MEDLINE
| ID: mdl-27755975
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 GENERALSIGNIFICANCE:
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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Archaea
/
Ácido Ditionitrobenzoico
/
Ferritinas
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article