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dc.contributor.authorCurrie, Marken
dc.date2010-08-31 16:28:35.76
dc.date2010-09-02 02:50:11.12
dc.date.accessioned2010-09-14T13:45:16Z
dc.date.available2010-09-14T13:45:16Z
dc.date.issued2010-09-14T13:45:16Z
dc.identifier.urihttp://hdl.handle.net/1974/6042
dc.descriptionThesis (Ph.D, Biochemistry) -- Queen's University, 2010-09-02 02:50:11.12en
dc.description.abstractCellulosomes are the most efficient plant cell wall degradation machines discovered to date. All cellulosomal components contain protein modules connected by linkers of varying lengths, which are predicted to be flexible. Consequently, structural studies of the cellulosome have employed a “dissect and build” strategy, whereby individual modules are studied in isolation with the hope to later model the intact complex. However, representative individual structures are now available for all of the cellulosome modules and many questions still remain. The studies described in this thesis depart from the ‘dissection’ stage and enter the ‘build’ stage of cellulosome structural studies of the cellulosome from Clostridium thermocellum. We first describe the crystal structure of a heterodimeric complex comprising the type-II cohesin (CohII) from cell surface anchoring protein SdbA and a trimodular C-terminal truncation of the CipA scaffoldin protein containing the ninth type-I cohesin module (CohI9), a linker, the X-module (X), and the type-II dockerin module (DocII). This structure revealed novel intertwining of scaffoldin molecules and extensive reciprocal contacts between the CohI9 and the X-module of another scaffoldin molecule. Sedimentation velocity experiments indicate dimerization also occurs in solution. We have carried out the crystallization and structural analysis of a heterotrimeric complex containing the CohI9—X-DocII:CohII complex bound to the type-I dockerin module (DocI) from the Cel9D enzyme, which represents the largest cellulosome fragment ever determined. Identical inter-scaffoldin interactions were observed in the heterotrimeric complex structure as were seen in the heterodimeric complex. However, small angle X-ray scattering (SAXS) studies indicate that this dimerization does not occur in solution. The crystal structures and additional SAXS studies reveal flexibility in the CohI9—X linker that is surprisingly restricted to two dimensions. In addition, this structure provides the first evidence of an orientation bias in DocI binding. Finally, SAXS was used to investigate modular orientations and linker flexibility in several cellulosome fragments. These studies indicate that cellulosomal linkers exhibit restricted and in some cases highly restrained flexibility. Specifically, scaffoldin linkers display two dimensional motions, enzymes maintain close contact with their cognate DocI modules, and enzyme positions rotate about 90° relative to neighbouring enzymes on the scaffoldin.en
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectCellulosomeen
dc.subjectX-Ray Crystallographyen
dc.subjectSAXSen
dc.titleSAXS and X-ray Crystallography Studies of the Cellulosome from Clostridium thermocellumen
dc.typethesisen
dc.description.degreePhDen
dc.contributor.supervisorJia, Zongchaoen
dc.contributor.departmentBiochemistryen
dc.degree.grantorQueen's University at Kingstonen


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