Thiazoline Ring Formation in an Engineered Intein Serves As a Splicing Switch

We have engineered an intein which autocatalytically forms a thiazoline ring at the N-terminal splicing junction and provided the first crystallographic evidence of this chemistry in an intein. The mutation that causes the aberrant thiazoline ring chemistry suggests that the highly conserved B-block Thr plays a pivotal role in orienting the resulting tetrahedral intermediate for resolution into a thioester, giving insight into the splicing mechanism. We demonstrate the stability of the thiazoline ring at mild conditions as well as sensitivity to hydrolytic opening of the ring under acidic conditions and high temperature. The reversibility of this thiazoline “trap” is of significant interest to biotechnological applications requiring a splicing activity switch.