Abstract 1271 Protein Splicing Activity of Two Inteins from Closely Related Thermophiles

Protein splicing is a post translational event in which the interrupting polypeptide in the precursor protein, known as the intein, is excised from the flanking polypeptides. In a self catalyzed reaction, the flanking polypeptides are ligated together resulting in a mature ligated product and an excised intein. My research project works with the thermophiles Thermococcus barophilus and Thermococcus kodakarensis. These two species have inteins that are similar in sequence but with different active site residues, and we are interested in how these differences affect protein splicing. Given that they are thermophiles, we have examined the impact of temperature on splicing activity. We hypothesized the in vitro splicing activity would be temperature dependent, and we observed optimal splicing at 50°C and minimal activity at 20°C. We explored the effects of mutating intein active site residues on the mechanism of splicing, in particular the third step of splicing which is Asn or Gln cyclization coupled to peptide bond cleavage. Given that T. barophilus is found in deep sea thermal vents and T. kodakarensis is a surface organism, we also hypothesize that pressure will affect splicing activity differently for their inteins. We thank the National Institutes of Health, NIGMS (Grant1R15GM132817-01) for supporting our research.