Synergistic Action of Crystallophore and Imaging‐Crystallophore Enhances the Production and Imaging of Protein Crystals

The synthesis and photophysical characterization of a new family of terbium(III) complexes called Imaging-crystallophore, substituted by π-conjugated antennas, is reported. Previous crystallophore variants (such as Tb-Xo4) have shown very interesting nucleating properties to obtain high-quality protein crystals. In addition, the Imaging-crystallophore emits in the green and accumulates in the protein crystals, enabling very easy crystal detection using fluorescence microscopy under one-photon but also under two-photon excitations. X-ray diffraction crystallography and molecular dynamics simulations demonstrate that the binding site of the Imaging-crystallophore in hen-egg white lysozyme (HEWL) is similar to that of the Tb-Xo4 but present additional hydrophobic interactions with the conjugated antennas. However, its nucleating properties are not as effective as those of Tb-Xo4. Finally, we emphasize the synergy between the two generations of crystallophore by preparing and analyzing the properties of the mix (10/0.2) corresponding to a mixture of Tb-Xo4 and Imaging-crystallophore. By combining the best of nucleating and imaging properties, we can easily obtain and detect high-quality protein crystals.