Positional effects of hydrophobic non-natural amino acid mutagenesis into the surface region of murine dihydrofolate reductase on enzyme properties

The ability to incorporate non-natural amino acids during protein biosynthesis has greatly broadened the amino acid sequence space available for protein engineering. The increasing number of non-natural amino acids provides novel sidechain chemistries, structures, and functionalities for engineered proteins including enzymes. Therefore, understanding how non-natural amino acid incorporation (non-natural amino acid mutagenesis) affects protein function is crucial. This study investigates the positional effects of hydrophobic non-natural amino acid mutagenesis of the enzyme surface on the enzyme structure and function with the ultimate goal of identification of permissive sites for non-natural amino acid mutagenesis. A bulky, hydrophobic non-natural amino acid, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at the solvent-exposed surface of a murine dihydrofolate reductase (mDHFR) enzyme. Incorporation of 2Nal was performed at six solvent-exposed sites (V43, E44, F142, E143, F179, and E180), generating six mDHFR variants. Incorporation of 2Nal into F142 or F179 did not exhibit any substantial change in the secondary structure and catalytic activities, whereas 2Nal incorporation at hydrophilic Glu sites significantly changed the secondary structure and catalytic activities. Such different responses upon 2Nal incorporation at hydrophobic and hydrophilic residues were likely due to the structural changes caused by the hydrophobicity change upon mutation. A non-conservative mutation of V43 with 2Nal significantly reduced the catalytic activities suggesting that a substantial size change of sidechain also causes the structural changes. These results suggest that even hydrophobic, bulky non-natural amino acids can be incorporated at the enzyme surface without compromising the enzymatic activities.