Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight |
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| Authors: | C Seth Pearson Reza Nemati Binbin Liu Jing Zhang Matteo Scalabrin Zhong Li Hongmin Li Dan Fabris Marlene Belfort Georges Belfort |
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| Institution: | 1. Howard P Isermann Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York;2. Department of Chemistry, State University of New York, Albany, New York;3. Laboratory of Computational & Structural Biology, Wadsworth Center, New York State Department of Health, Albany, New York;4. Department of Biomedical Sciences, School of Public Health, Albany, State University of New York, Albany, New York |
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| Abstract: | We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing. |
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| Keywords: | cleavage control molecular switch protein splicing thiazoline crystal structure |
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