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dc.contributor.advisorHefty, P. Scott
dc.contributor.authorKemege, Kyle Evan
dc.date.accessioned2013-09-30T20:05:31Z
dc.date.available2013-09-30T20:05:31Z
dc.date.issued2013-05-31
dc.date.submitted2013
dc.identifier.otherhttp://dissertations.umi.com/ku:12739
dc.identifier.urihttp://hdl.handle.net/1808/12328
dc.description.abstractInfections by chlamydial species are of significant impact to global public health, causing sexually transmitted infections, blinding trachoma and pneumonia. Despite its importance, there are many aspects of chlamydial biology that are not completely understood, including the mechanisms by which it infects, persists and replicates in its host cells. The reason for this ignorance of basic chlamydial biological processes is because there is an abundance of Open Reading Frames (ORFs) of unknown function present in chlamydial genomes, almost 30% of the entire genome in many species. This is likely due to the relatively large phylogenetic distance between Chlamydiae and better-understood bacteria such as E. coli and B. subtilis. Current strategies of genome annotation rely on the presence of homology to genes of known function and these approaches have not been effective in annotating chlamydial genomes. In an effort gain insight into the function of these chlamydial ORFs of unknown function, I utilized structural information (both computational and experimentally derived) about three proteins of interest. Based on these structural studies, hypotheses concerning the functions of these proteins were formed and then tested. Together, my findings not only provide valuable information about these proteins of unknown function, but they also serve to demonstrate both the strengths and shortcomings of the overall approach of utilizing structural information for functional prediction. One example of this approach is my work on the chlamydial ORF CT296. Although this protein was annotated as having an unknown function (due to insignificant homology to proteins of known function), it had been experimentally characterized as an iron-dependent transcription factor. Having an experimentally characterized function allowed me to test my approach of utilizing structural information to predict function on a protein with a relatively well-understood function. Surprisingly, structural information of this protein suggested that it functions as a Fe(II) 2-oxoglutarate-dependent enzyme and not as a transcription factor. Subsequent functional analyses of the protein were unable to reproduce previous reports of its DNA binding. Together, my findings suggest that this protein may not function as a transcription factor. A second example of my structure-function approach was applied to the chlamydial protein CT584. This protein was first experimentally described as interacting with the chlamydial Type Three Secretion System (T3SS) needle protein in an interactome study. This observation, combined with a subsequent biophysical characterization of the protein lead to an initial hypothesis that the protein may be a chlamydial T3SS needle tip protein. However, results of structural studies on the protein reveal a structure that is not similar to any of the known T3SS needle tip proteins. Additionally, functional studies on the protein focusing on identifying its localization on chlamydial organisms revealed localization patterns not consistent with its proposed role as a T3SS needle protein. Together, my studies suggest that this protein may not function as a needle tip protein. A final example of the utility of structural information for informing function concerns chlamydial ORF CT009. This protein was annotated in many chlamydial species as a protein of unknown function; however, bioinformatics analyses had identified it as a helix-turn-helix containing transcription factor. Results of computational and experimental structures of this protein show structural similarity to the protein RodZ, a key component of the bacterial morphogenic complex. Subsequent functional analyses of CT009 demonstrate that this protein has the characteristics of a chlamydial homolog to RodZ.
dc.format.extent206 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectBiochemistry
dc.subjectMolecular biology
dc.subjectMicrobiology
dc.subjectChlamydia
dc.subjectCrystallography
dc.subjectProtein
dc.titleSTRUCTURAL AND FUNCTIONAL STUDIES OF THREE PROTEINS OF UNKNOWN FUNCTION ENCODED BY CHLAMYDIA TRACHOMATIS
dc.typeDissertation
dc.contributor.cmtememberLamb, Audrey
dc.contributor.cmtememberEgan, Susan
dc.contributor.cmtememberAzuma, Yoshiaki
dc.contributor.cmtememberDe Guzman, Roberto N.
dc.contributor.cmtememberScott, Emily E.
dc.thesis.degreeDisciplineMolecular Biosciences
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
dc.rights.accessrightsopenAccess


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