Fig 1.
Fluorescence spectra of HFt-LBT Tb(III) complex.
Fluorescence spectra of HFt-LBT Tb(III) (red line) and wild type mouse HFtTb(III) complexes (blue line) at the same protein concentration (1 μM monomer). The main emission band due to the 5D4-7F5 (Tb d-f orbitals) electronic transition at 544 nm is accompanied by the weaker 5D4-7F6 transition. Spectra were recorded after protein saturation with TbCl3 in 0.1 M MES buffer pH 6.4 (see Methods).
Fig 2.
Fluorescence titration of HFt-LBT with Tb(III).
A) Fluorescence titration of HFt-LBT (1μM monomer) with incremental concentration of Tb(III) in 0.1 M MES buffer pH 6.4. Emission spectra were recorded in 1 cm pathlength cuvette upon excitation at 295 nm. B) Fluorescence intensity of HFt-LBT Tb(III) complex as a function of the Tb(III)/HFt-LBT ratio. Fluorescent intensity was recorded at 545 nm, corrected for dilution factor and normalized to the emission maximum.
Fig 3.
Terbium binding sites from X-ray crystallography.
A) An overall view of the 24-meric shell of HFt-LBT Tb(III) showing the positions of Tb(III), displayed as spheres, is shown. In the close-up view, the omit map contoured at 3 σ as a green mesh is shown for the B) ferroxidase center where Tb(III) is depicted in magenta and for the C) 3-fold axes where Tb(III) is depicted as a yellow sphere and the residues Glu131 and Glu134 of three different monomers are depicted as sticks in light blue, cyan and orange, respectively.
Fig 4.
Cryo-electron microscopy structure of HFt-LBT.
Internal view, sliced at the protein center, of the density map of HFt-LBT Tb(III) (resolution = 7.1 Å). The crystallographic model of HFt-LBT Tb(III) (purple ribbons) was rigid-body fitted into the Cryo-EM map (gray solid) using USCF Chimera [50]. The E helices at the C-terminal part of each monomer are highlighted in blue. LBT tails fused to the E helices are enlarged on the right panels showing the top view (dashed line box) and the side-view (continuous line box). Well-defined electron density extends over the end of the X-ray model as due to the presence of the terminal LBT tails and it approximately covers 14 Å distance, that corresponds to the length of 6–7 amino acids (main chain). Scale bar = 10 Å.
Fig 5.
Confocal microscopy images and flow cytometry analysis of HFt-LBT Tb(III) uptake by selected tumor cell lines.
DU-145, HCT-116, MDA-MB-231 and SKOV-3 cancer cells were incubated with either HFt-LBT Tb(III) or wild type mouse HFt (0.5 mg/ml) for 60 min. A) Images acquired by confocal microscopy showing side by side comparison of cellular distribution of HFt-LBT Tb(III) and HFt conjugated with FITC. Scale bar = 50 μm. B) Flow cytometry analysis of HFt-LBT Tb(III) and HFt cellular uptake.