Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A

L alpha-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal L beta-, D alpha-, and D beta-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomercontaining proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, L alpha-Asp at position 121 in RNase A was replaced by L beta-, D alpha-, and D beta-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with L alpha-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of L alpha-, D alpha-, and D beta-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.