Probing bulky ligand entry in engineered archaeal ferritins

Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):450-456. doi: 10.1016/j.bbagen.2016.10.007. Epub 2016 Oct 15.

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.

Keywords: Binding kinetics; Ferritin; Nano-scaffold; Self-assembly.

MeSH terms

  • Archaea / genetics
  • Archaea / metabolism*
  • Archaeoglobus fulgidus / genetics
  • Archaeoglobus fulgidus / metabolism
  • Cysteine / metabolism
  • Dithionitrobenzoic Acid / metabolism*
  • Ferritins / genetics
  • Ferritins / metabolism*
  • Kinetics
  • Ligands
  • Mutation / genetics
  • Pyrococcus furiosus / genetics
  • Pyrococcus furiosus / metabolism
  • Sulfhydryl Compounds / metabolism

Substances

  • Ligands
  • Sulfhydryl Compounds
  • Ferritins
  • Dithionitrobenzoic Acid
  • Cysteine