Fig. 5: Crystal structure of dIG8-CC and functional loop scaffolding.

a Design model of dIG8-CC with a disulfide bridge (spheres) between β-strands 3 and 6. b SEC-MALS analysis of dIG8-CC estimates a molecular weight between monomer (8.3 kDa) and dimer (16.6 kDa). c Design model (green) in comparison with the crystal structure with PDB accession code 7SKP (gray, chain C). d Cross-β motif connections and core sidechain interactions in the design and the crystal structure. The β-arch helix and loop conformations are well preserved across monomer copies in the crystal asymmetric units (insets). e Crystal homodimer interface by parallel pairing between the two terminal β-strands, which are stabilized through hydrophobic and salt-bridge interactions (inset). f Computational model of dIG8-CC with a grafted EF-hand motif (design EF61_dIG8-CC, cartoon), showing Tb³⁺ (sphere) bound to EF-hand motif residues (sticks). Tb³⁺ luminescence is sensitized by absorption of light (purple) by a proximal tyrosine residue on the EF-hand motif with subsequent fluorescence resonance energy transfer (FRET) to Tb³⁺, resulting in Tb³⁺ luminescence (green). g Far-ultraviolet circular dichroism spectra of EF61_dIG8-CC without Tb³⁺ (blue: 25 °C; green: 55 °C; red: 75 °C; black: 95 °C). h Time-resolved luminescence emission spectra in 100 μM Tb³⁺ final concentrations for EF61_dIG8-CC (blue) and dIG8-CC (red) at 20 µM. Time-resolved luminescence intensity is given in relative fluorescence units (RFU). i Tb³⁺ concentration-dependent time-resolved luminescence intensity of 20 µM EF61_dIG8-CC using excitation wavelength λ_ex  = 280 nm and emission wavelength λ_em  = 544 nm. Normalized intensities are fit to a one-site binding model by non-linear least squares regression (K_d = 267 μM).