Computational design of a homotrimeric metalloprotein with a trisbipyridyl core

Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2'-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)₃]²⁺ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications.