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Investigation of prokaryotic immune defense system with quantitative and structural mass spectrometry

dc.contributor.advisorUrlaub, Henning Prof. Dr.
dc.contributor.authorSharma, Kundan
dc.titleInvestigation of prokaryotic immune defense system with quantitative and structural mass spectrometryde
dc.contributor.refereeStülke, Jörg Prof. Dr.
dc.description.abstractengBacteria and archaea have evolved an adaptive and heritable immune defense system comprising a CRISPR (clustered regularly interspaced short palindromic repeats) locus and Cas (CRISPR-associated) proteins that targets mobile genetic elements such as phages and plasmids. The molecular memory of previous infections is transcribed into crRNAs (CRISPR RNAs) which serve as a template to guide the hydrolysis of incoming foreign genetic material. The CRISPR-Cas system is divided into three Types (I, II and III) on the basis of signature cas genes and further subtypes defined by the protein machinery and target specificity. During my PhD work, I investigated the Type I and Type III CRISPR-Cas systems using quantitative and structural mass spectrometry approaches.  The first part of this work focused on using relative quantitative approach to study the effect of a cas gene deletion on the expression levels of other Cas proteins in Haloferax volcanii. A comparison between the proteomes of H. volcanii wild type and deletion strains using stable-isotope dimethyl labeling showed that the removal of a cas gene is compensated by an overall increase in the expression of cas gene cluster. In addition, the absolute amounts of Cas protein components in multi-subunit Cascade complexes from H. volcanii and Clostridium thermocellum were determined using intensity based absolute quantification. The results were used to determine the stoichiometry of Cas proteins in these multi-subunit protein complexes which is valuable for the further investigation of molecular interactions within these complexes.  Further in this work, the UV induced protein-RNA cross-liking approach was utilized to investigate RNA binding regions in single (recombinant) Cas proteins such as the archaeal and bacterial Cas6b proteins and the Cas7 family proteins from four different organisms. These structural studies were also extended to multi-subunit crRNP complexes such as the Type I-E Cascade complex from E. coli and Type III-A Csm complex from Thermus thermophilus. The information derived from the cross-linking studies could validate several protein-RNA interactions reported earlier in X-ray crystallography studies. In addition to identifying new RNA binding regions in the Cas proteins, the identified cross-links could also be mapped to conserved regions of these proteins within modified RNA binding domains. The results provided unbiased evidence of direct protein-RNA interaction in in vitro and in vivo purified crRNP complexes. Lastly, a lysine directed chemical cross-linking approach is presented for the investigation of protein-protein interactions between different Cas proteins in the C. thermocellum Cascade complex where more than 126 inter-protein interactions were identified. These results constitute the first step towards MS based structural modeling of crRNP
dc.contributor.coRefereeRehling, Peter Prof. Dr.
dc.contributor.thirdRefereeCramer, Patrick Prof. Dr.
dc.contributor.thirdRefereeGroß, Uwe Prof. Dr.
dc.contributor.thirdRefereeDaniel, Rolf Prof. Dr.
dc.subject.engProkaryotic Immune Defensede
dc.subject.engMass Spectrometryde
dc.subject.engStructural proteomicsde
dc.subject.engProtein-RNA cross-linkingde
dc.subject.engProtein-Protein cross-linkingde
dc.subject.engQuantitative proteomicsde
dc.subject.engDimethyl labelingde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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