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STRUCTURE-FUNCTION INSIGHTS INTO THE BIOCHEMICAL PROPERTIES AND PHOSPHOLIPID BILAYER INTERACTIONS OF THE SAPOSIN-LIKE DOMAIN OF PLANT ASPARTIC PROTEASES

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Title: STRUCTURE-FUNCTION INSIGHTS INTO THE BIOCHEMICAL PROPERTIES AND PHOSPHOLIPID BILAYER INTERACTIONS OF THE SAPOSIN-LIKE DOMAIN OF PLANT ASPARTIC PROTEASES
Author: Bryksa, Brian Charles
Department: Department of Food Science
Program: Food Science
Advisor: Yada, Rickey Y.Marangoni, Alejandro
Abstract: This 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.
URI: http://hdl.handle.net/10214/10177
Date: 2016-12
Rights: Attribution-NoDerivs 2.5 Canada


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Attribution-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NoDerivs 2.5 Canada