Stabilization of hyperactive dihydrofolate reductase by cyanocysteine-mediated backbone cyclization

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.