Studies on the structure and composition of the outer membrane of Caulobacter crescentus
department or school: Science, Faculty of
department or school: Microbiology and Immunology, Department of
data provider: DSpace
degree campus: UBCV
creator: Jones, Michael D.
issued: 2015-10-24T07:27:24
issued: 2015
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: Classically, outer membranes are half lipid, half protein, and the outmost layers of Gram- negative bacteria. For Caulobacter crescentus the outer membrane is the penultimate layer beneath a protein surface layer (S-layer). The S-layer of the caulobacter cell envelope is an exciting platform for high density peptide display and biotechnology development. We focused on elucidating the structure of the outer membrane by crystallizing the S-layer protein, RsaA; solving the structure of the the lipopolysaccharide; and characterizing a newly discovered porin, OmpW. S-layer proteins are highly resistant to crystallization, because wo-dimensional S-layer formation out competes three-dimensional crystal formation. To achieve a crystallisable form of RsaA, a C-terminal truncation version was constructed and expressed in the native host, C. crescentus. The secreted protein was prone to aggregation, so low agitation and slow concentration protocols had to be developed. The RsaA truncate produced large crystals that diffracted to <2.5 Å. Solving the phases proved to be a serious hurdle and the final protein structure remains unsolved. The lipopolysaccharide of C. crescentus is the anchor that supports the S-layer. The structure of the lipid A portion was solved previously but structures for the core oligosaccharide and the O-polysaccharide had not been deduced. In collaboration with Dr. Evgeny Vinogradov, these remaining structures were solved. The core oligosaccharide has a branched heptasaccharide structure. The O-polysaccharide is a heptasaccharide containing the dideoxy sugar N-acetylperosamine. Additionally, a rhamnan polysaccharide was discovered and its structure was determined. Porins, non-specific passive protein channels, are significant components of classical Gram-negative outer membranes. Despite this, no porin had ever been identified in C. crescentus. We report the identification and characterization of the porin OmpW in C. crescentus. OmpW has low conductance of…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/55040
note: Studies on the structure and composition of the outermembrane of Caulobacter crescentusbyMichael D JonesB. Science, Specialization in Biotechnology, University of Alberta, 2006M. Science, Pharmaceutical Sciences, University of Alberta, 2008A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDoctor of PhilosophyinTHE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES(Microbiology and Immunology)The University of British Columbia(Vancouver)October 2015c©Michael D Jones, 2015AbstractClassically, outer membranes are half lipid, half protein, and the outmost layers of Gram-negative bacteria. For Caulobacter crescentus the outer membrane is the penultimate layerbeneath a protein surface layer (S-layer). The S-layer of the caulobacter cell envelope is anexciting platform for high density peptide display and biotechnology development. We focusedon elucidating the structure of the outer membrane by crystallizing the S-layer protein, RsaA;solving the structure of the the lipopolysaccharide; and characterizing a newly discovered porin,OmpW.S-layer proteins are highly resistant to crystallization, because wo-dimensional S-layerformation out competes three-dimensional crystal formation. To achieve a crystallisable formof RsaA, a C-terminal truncation version was constructed and expressed in the native host,C. crescentus. The secreted protein was prone to aggregation, so low agitation and slowconcentration protocols had to be developed. The RsaA truncate produced large crystals thatdiffracted to <2.5 Å. Solving the phases proved to be a serious hurdle and the final proteinstructure remains unsolved.The lipopolysaccharide of C. crescentus is the anchor that supports the S-layer. The struc-ture of the lipid A portion was solved previously but structures for the core oligosaccharideand the O-polysaccharide had not been deduced. In collaboration with Dr. Evgeny Vinogradov,these remaining structures were solved. The core oligosaccharide has a branched heptasac-char…
has type: Thesis/Dissertation
date issued: 2015-11
is shown at: 10.14288/1.0166778
language: eng
degree discipline: Microbiology and Immunology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-ShareAlike 2.5 Canada
rights URI: http://creativecommons.org/licenses/by-sa/2.5/ca/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/55040