A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe, Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C-alpha/beta positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D2O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62 - 79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.