Abstract
Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation.
Highlights
Ferritin is a cage-like protein made of 24 subunits arranged in octahedral 432 symmetry with an outer diameter of roughly 12 nm and an inner diameter of 8 nm [1,2]
The titration endpoint was reached at 1.7 equivalent amounts of Tb(III) per subunit instead of the predicted 1 equivalent based on the presence of one lanthanide binding tag (LBT) moiety per monomer
The LBT has six metal-binding residues that form a coordination sphere around a lanthanide (III) ion and is characterized by high affinity and strong Forster resonance energy transfer (FRET) effect from the tag encoded tryptophan residue to the Terbium or Europium ions
Summary
Ferritin is a cage-like protein made of 24 subunits arranged in octahedral 432 symmetry with an outer diameter of roughly 12 nm and an inner diameter of 8 nm [1,2]. The symmetrical positioning of three or four subunits in the protein shell results in the formation of eight. The funder provided support in the form of salaries for authors Trabuco, MC, who is employed at Molirom as a Marie Curie Fellow within the framework of the Xprobe EU project, and Taciak B., Rygel T and Krol M., who are employed at Cellis within the framework of the National Science Center of Poland. The specific roles of these authors are articulated in the ‘author contributions’ section
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