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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorBelfort, Georges
dc.contributorBelfort, Marlene
dc.contributorKarande, Pankaj
dc.contributorShekhtman, Alexander
dc.contributor.authorPearson, C. Seth
dc.date.accessioned2021-11-03T09:00:26Z
dc.date.available2021-11-03T09:00:26Z
dc.date.created2018-07-27T15:08:39Z
dc.date.issued2018-05
dc.identifier.urihttps://hdl.handle.net/20.500.13015/2198
dc.descriptionMay 2018
dc.descriptionSchool of Engineering
dc.description.abstractOur work focuses on two of the three inteins found in the human pathogen Mycobacterium tuberculosis (Mtu), RecA and SufB. The intein-interrupted proteins are important for the viability of Mtu. Preventing intein excision, and thus the formation of important functional proteins, suggests that in vivo intein inhibition may be used as an anti-mycobacterial strategy. Building upon previous work with the chemotherapeutic cisplatin, we demonstrated potent in vitro inhibition of the RecA intein with novel cisplatin analogs using a fluorescent split-GFP assay. We also provided the structure of an intein-Pt co-crystal containing a platinum adduct at each catalytic cysteine, and calculate the binding kinetics using NMR. Perhaps most interestingly, we showed that the cisplatin ligands required for DNA binding are not present when bound to the intein, suggesting that less cross-reactive, and therefore less toxic, cisplatin analogs may be identified.
dc.description.abstractDuring this process, the intein breaks two peptide bonds and forms a third. Taking advantage of the intein’s ability to catalyze these reactions, both within the natural host as well as in vitro for biotechnological applications, provides an attractive opportunity. Implicit in effective utilization is an ability to control when, and if, catalysis occurs. Because inteins require no cofactors and have evolved an exquisite spatial and temporal coordination between the various peptide- making and -breaking reactions, achieving such control is not trivial. The work presented here aims to investigate how intein activity can be controlled in several disparate contexts.
dc.description.abstractThe final aspect of our work is based on the intriguing hypothesis that inteins are not simply parasitic elements, but offer a beneficial protein-level control of host protein function. We demonstrated that Mtu SufB activity is inhibited in vivo by oxidizing agents and copper ions, stressors to which the mature host protein is exquisitely sensitive. We provided evidence using ICP-MS and SDS-PAGE gel shift assays that suggest that the inhibition by copper ions is not direct, but rather part of a redox control mechanism involving oxidation of the catalytic cysteines. Further work to test this hypothesis must be performed and several lines of experimentation are proposed.
dc.description.abstractTurning to methods of control for in vitro applications, we introduced a mutation in Mtu RecA that causes the intein to spontaneously form a thiazoline ring at the N-terminal splicing junction. While present, the thiazoline ring prevents any further intein chemistry. We provided the first crystallographic evidence at 1.54 Å resolution of this aberrant thiazoline chemistry in an intein. The crystal structure provides evidence for the existence of a tetrahedral intermediate resulting from nucleophilic attack by Cys1 on the adjacent extein residue carbonyl carbon. Additionally, the mutation that results in the thiazoline ring suggests that a highly conserved B-block Thr plays a pivotal role not only in “spring-loading” the scissile N-terminal peptide bond, but also in orienting the resulting tetrahedral intermediate for resolution into a thioester required for splicing, giving insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic opening of the ring under acidic conditions and increased temperature using mass spectrometry. A pH cycling strategy to induce N-terminal cleavage is provided, which may be of interest for biotechnological applications requiring a splicing activity switch such as single-step affinity-based purifications.
dc.description.abstractInteins are protein elements that autocatalytically excise themselves from a host protein in a process called protein splicing. The two flanking sequences of the host protein, called the N- and C-exteins, are subsequently ligated with a native peptide bond, resulting in a mature protein.
dc.description.abstractOverall, this work investigates strategies for controlling inteins in the realms of drug development, protein purification, and host regulation and stress response.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectChemical engineering
dc.titleInteins as biomolecular switches : applications in drug discovery, protein purification, and stress response
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid179004
dc.digitool.pid179005
dc.digitool.pid179006
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Chemical and Biological Engineering


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