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dc.contributor.advisorSarma, Siddhartha P
dc.contributor.authorRami, Mihir
dc.date.accessioned2024-02-29T06:07:09Z
dc.date.available2024-02-29T06:07:09Z
dc.date.submitted2024
dc.identifier.urihttps://etd.iisc.ac.in/handle/2005/6424
dc.description.abstractThis thesis involves a detailed structural study of two molecular systems, a small disulfide-rich peptide LL-TIL and a large catalytic subunit of AHAS. Subtilisin inhibitors play an important role in fighting against these harmful microorganisms. LL-TIL, found in skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays established that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided a detailed view of the structural basis of inhibition. The energy calculation by MM-PBSA analysis for the LL-TIL-subtilisin complex predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL was generated, which broadened the inhibitory profile and attenuated subtilisin inhibition by two orders of magnitude. These results provide a valuable template to engineer more specific and potent TIL-type subtilisin inhibitors. AHAS catalyzes the first step in the biosynthetic pathway of branched-chain amino acids. The active site resides within the interface of the independently folded catalytic domains α and γ. The α domain of the catalytic subunit of E. coli AHAS I (ilvBα) aggregated at high concentrations required for NMR. A new approach was designed involving the study of the α and γ domains from T. maritima (TmCSUα and TmCSUγ). The domains were found to be tethered by a disulfide bond. Domain-specific isotopically labelled samples were successfully prepared for the NMR study. However, the high-resolution multidimensional NMR experiments suffered from low sensitivity, which hindered the sequence-specific assignments.en_US
dc.language.isoen_USen_US
dc.relation.ispartofseries;ET00436
dc.rightsI grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertationen_US
dc.subjectNMR Spectroscopyen_US
dc.subjectStructural Biologyen_US
dc.subjectProtease inhibitoren_US
dc.subjectEnzymesen_US
dc.subjectPeptideen_US
dc.subjectLL-TILen_US
dc.subject.classificationResearch Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Molecular biologyen_US
dc.titleSolution NMR studies of a potent subtilisin inhibitor from Budgett’s frog and the catalytic subunit of AHASen_US
dc.typeThesisen_US
dc.degree.namePhDen_US
dc.degree.levelDoctoralen_US
dc.degree.grantorIndian Institute of Scienceen_US
dc.degree.disciplineFaculty of Scienceen_US


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