Structure-function insights into the biochemical properties and phospholipid bilayer interactions of the saposin-like domain of plant aspartic proteases

dc.contributor.advisorYada, Rickey Y.
dc.contributor.advisorMarangoni, Alejandro
dc.contributor.authorBryksa, Brian Charles
dc.date.accessioned2017-01-05T21:21:36Z
dc.date.available2017-01-05T21:21:36Z
dc.date.copyright2016-12
dc.date.created2016-06-23
dc.date.issued2017-01-05
dc.degree.departmentDepartment of Food Scienceen_US
dc.degree.grantorUniversity of Guelphen_US
dc.degree.nameDoctor of Philosophyen_US
dc.degree.programmeFood Scienceen_US
dc.description.abstractThis thesis is an investigation of structure-function relationships of the saposin-like domain of plant aspartic proteases. Many plant aspartic proteases contain an additional sequence of approximately 100 amino acids termed the plant-specific insert which is involved in host defense and vacuolar targeting. Similar to all other saposin-like proteins, the plant-specific insert functions via protein-membrane interactions, however, the structural basis for such interactions have not been studied and the nature of plant-specific insert-mediated membrane disruption have not been characterized. This thesis presents the first comprehensive structure-function investigation of the less-understood arm of the saposin-like protein family, the so-called “swaposins”. Among the findings presented here are the quaternary, tertiary and secondary structures of the plant-specific insert from Solanum tuberosum (potato) aspartic proteinase both in terms of pH and lipid bilayer presence, the identification of a structure in potato saposin (Ile1-Leu20) that is a universal membrane penetrating motif based upon structural alignment, delineation of the structural basis for the acid pH requirement for bilayer interaction (pH-sensitive dimerization) and positively charged point of contact for anionic bilayers (Lys83) located at the C-terminal end of helix 3, a positively charged residue within an uncommon anti-bilayer motif found in some flocculant proteins and spider silk structural proteins, among others. Atomic force microscopy revealed that potato plant-specific insert destabilized (softened) bilayer whereas cryo-transmission electron microscopy showed several distinct shapes induced in LUV’s upon addition of potato saposin. Lastly, comparative characterizations of potato plant-specific insert along with three other plant saposin-like domains from barley (Hordeum vulgare), flowers of Cardoon thistle (Cynara cardunculus L.) and Rockcress (Arabidopsis thaliana) were carried out revealing that reduction of the disulfide bonds of potato swaposin caused a drastic increase in bilayer fusion rate and increase in typical fusion product sizes. Arabidopsis swaposin with reduced cystines showed relatively minor alterations to its fusion profile while essentially no difference to the fusogenic activities of barley and Cardoon swaposins were discernable upon reduction of their disulfide bonds. Taken together, implications for swaposin mechanism of action as well as future research directions are discussed.en_US
dc.description.sponsorshipNatural Sciences and Engineering Research Council of Canada
dc.description.sponsorshipCanada Research Chairs program
dc.identifier.urihttp://hdl.handle.net/10214/10177
dc.language.isoenen_US
dc.publisherUniversity of Guelphen_US
dc.rightsAttribution-NoDerivs 2.5 Canada*
dc.rights.urihttp://creativecommons.org/licenses/by-nd/2.5/ca/*
dc.subjectsaposinen_US
dc.subjectbilayer fusionen_US
dc.subjectvesicle leakageen_US
dc.subjectswaposinen_US
dc.subjectplant-specific inserten_US
dc.subjectaspartic proteaseen_US
dc.subjectsaposin-like domainen_US
dc.subjectplant-specific sequenceen_US
dc.subjectplant immune responseen_US
dc.subjectfusogenicen_US
dc.subjectvacuolar targettingen_US
dc.titleStructure-function insights into the biochemical properties and phospholipid bilayer interactions of the saposin-like domain of plant aspartic proteasesen_US
dc.typeThesisen_US

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