Developing a 4-Helix Bundle Protein Engineering Platform for Isolating Rare-Earth Elements

Rare-earth elements (REEs) are crucial for sustainable industrial processes, but current extraction methods are environmentally damaging. Our research explores using proteins that bind REEs as a potentially eco-friendly alternative for purification. Lanmodulin (LanM) is a natural protein known for its extremely strong (picomolar) affinity for REEs. However, LanM's disordered structure makes it challenging for engineering. To overcome this, we developed a protein engineering platform based on the stable 4-Helix Bundle (4HB) framework, which has 3 helix-loop-helix regions. We incorporated the core REE-binding segments, a helix-loop-helix region, from native LanM (specifically, LanM loop1 into the 4HB's second loop) in prearranged optimal REE-binding configuration. The 4HB platform underwent engineering to eliminate off-target REE binding as well and was then rationally designed for specificity towards REEs with smaller ionic radii. This resulted in successful REE binding with nanomolar affinities. Furthermore, we employed machine learning and molecular dynamics to customize REE-binding loops for individual REE selectivity. Crucially, we demonstrated that the 4HB framework is reusable and capable of binding and releasing REEs under mild, environmentally friendly conditions.