Disulfide Engineering Catches a Self-Splicing Intein In a Redox Trap

Inteins are autocatalytic proteins with broad biotechnological appeal.  They self-splice from internal positions of precursor polypeptides in all domains of life.  We report here the design of a redox-sensing intein and its use to enable both basic and applied studies of these elements.  Redox regulation was achieved by engineering a disulfide bond between the intein’s catalytic cysteine residue and a cysteine residue substituted in the flanking N-terminal “extein” sequence.  We show that this intein’s activity can be turned on in vitro by the addition of a reducing agent, thereby providing an efficient system for expressed protein ligation (EPL).  Second, we present the crystal structure of the redox-trapped intein, which includes the first high resolution view of a transient, and conformationally distorted, intein-extein junction. In addition to facilitating structure-activity studies of inteins, this work motivates speculation about similar regulatory mechanisms that may exist for inteins or related autoprocessing proteins in nature.