(672c) Interrogating Thiazoline Ring Formation in an Engineered Reca Intein
Inteins are naturally occurring protein elements that autocatalytically excise themselves from a non-functional precursor and ligate the flanking protein segments with a peptide bond, resulting in a functional protein. The ability of inteins to perform cleavage and formation of peptide bonds has made inteins attractive for biotechnology applications. Native and engineered inteins have been utilized for purification (Wood 1999, 2005), site specific protein labeling (Muralidharan 2006), and even redox sensing (Callahan 2013). Though the overall steps of protein splicing are understood, atomic details have not been fully elucidated. The ability to engineer useful inteins depends on these atomic details of the role of all conserved residues in the splicing site.
Here, we investigate an engineered RecA intein that readily forms a thiazoline ring between the first cysteine of the intein and the preceding N-extein residue, likely the result of dehydration of an oxythiazolidine anion intermediate in the first step of protein splicing. We investigate the stability of this ring through crystallographic and mutational studies. The mechanistic insights gained from these investigations may assist in the design of tunable inteins with a variety of biotechnological applications.