Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins

We report the rational construction of de novo-designed biliverdin-binding proteins by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor autocatalytically in vitro, like photosensory proteins that bind BV (and related bilins or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identification of a suitable site for ligation of the cofactor to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to inform the structure-function relationships of the cofactor binding pocket. Holoprotein formation stabilized BV, resulting in increased far-red BV fluorescence. Via removal of segments extraneous to cofactor stabilization or bundle stability, the initial 15 kDa de novo-designed fluorescence-activating protein was truncated without any...