Crystal Structure of a Kinetically Persistent Transition State in a Computationally Designed Protein Bottle

The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Herein we use packing interactions within the core of a protein to stabilize the planar TS for rotation around the central C-C bond of biphenyl so that it can be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis we identified a protein in which the side chain of p-biphenylalanine is kinetically trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.