Abstact

Lanthanide-protein scaffolds hold significant promise for the design of functional biomaterials. Yet the selective incorporation of multiple lanthanide ions with distinct properties into discrete sites at tuneable distances within a single construct remains a key challenge. Here, we report the rational design and structural characterization of the first de novo coiled coil capable of binding two different lanthanide ions at independent, non-equivalent sites with defined intermetallic spacing. By installing orthogonal coordination environments, comprising Asn(3)Asp(3) and Asp(3)-only motifs, at defined positions along the coiled coil axis, we achieve precise, site-specific metal binding across a series of constructs spanning 1 to 5 nm. Site occupancy and intermetallic distances were validated using luminescence, electron paramagnetic resonance (EPR) spectroscopy, mass spectrometry and X-ray crystallography. The latter reveals the first structure of a coiled coil bound to two Tb(3+) ions, and the shortest non-bridged metal-metal distance reported to date in such a scaffold (11.9 A). The chemically distinct coordination sites enable sequential and selective metal loading. Remarkably, this system is capable of binding two different lanthanides, Tb(3+) and Yb(3+), at distinct sites, despite their extremely similar coordination chemistries. These results establish a robust and modular platform for constructing nanometre-scale molecular rulers, and highlight new avenues for the rational design of multifunctional metalloproteins.