Abstract
Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. The intein that interrupts the Pyrococcus abyssi PolII protein can be purified as part of a precursor fusion protein. Splicing can be initiated by an increase in temperature, which allows for detailed kinetic analysis of the overall splicing process as well as the individual steps. We have studied the influence of the residue immediately upstream of the intein on the rate of the first step of splicing, an amide to thioester rearrangement. Modifications leading to changes of that rate can result in splicing side reactions. We also have examined the influence of residues constituting a putative oxyanion hole on the rate of the first step of the reaction and the extent of splicing. Finally, we present data on the influence of two conserved downstream His residues on the rate of step three of splicing, Asn cyclization coupled to peptide bond cleavage.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
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