Unexpected diastereoselective chemistry on a 2D protein surface

Enantioselective enzymes feature structured catalytic sites positioned within binding pockets. In contrast, widely occurring flat protein surfaces, which have been suggested to have emerged early in ancestral protein evolution as solvent-exposed β sheets, appear unlikely to catalyze asymmetric reactions because they lack the necessary scaffold for interacting with substrates in three dimensions. At the near-flat, water-accessible surface within the α-hemolysin transmembrane pore, we now demonstrate remarkable diastereoselectivity in hemithioacetal formation between a cysteine side chain and a series of aldehyde substrates. After protein surface remodeling by mutagenesis, diastereomeric ratios of up to 95:5 (kinetic control) and 98:2 (thermodynamic control) were achieved with a range of aromatic aldehydes. Molecular dynamics simulations confirmed asymmetric interactions between adducts and nearby side chains in a two-dimensional plane. Our findings indicate that flat protein surfaces can scaffold stereoselective chemistry, thereby expanding the designable protein space for catalyst engineering and providing insight into the origin of selective enzymes.