Dataset Information

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

ABSTRACT: Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein "zymogens" can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems.

SUBMITTER: Gramespacher JA

PROVIDER: S-EPMC5533455 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Publications

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

Gramespacher Josef A JA Stevens Adam J AJ Nguyen Duy P DP Chin Jason W JW Muir Tom W TW

Journal of the American Chemical Society 20170607 24

Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein "zymogens" can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacteria ...[more]

PMID: 28562027

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Project description:Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.

Dataset Information

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

Publications

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

Similar Datasets

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