Hayley, Michael (2007) Biochemical analysis of toposome, a protein mediating membrane-membrane interactions in the sea urchin egg & embryo. Doctoral (PhD) thesis, Memorial University of Newfoundland.
- Accepted Version
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The yolk granule is the most abundant membrane-bound organelle present in sea urchin eggs and embryos. The major protein component of this organelle, major yolk protein/toposome, accounts for approximately 50% of the total yolk protein and has also been shown to be localized to the embryonic cell surface. Biochemical and cell biological analysis in several laboratories have defined a role for toposome in mediating membrane-membrane interactions. -- In this study we have examined calcium-toposome interaction. Increasing concentrations of calcium resulted in an increase in alpha helical content from 3.0 to 22.0%, which occurred with an apparent dissociation constant (calcium) of 25 μM. In parallel experiments, toposome binding to liposomes required similar concentrations of calcium; an apparent dissociation constant (calcium) of 25 μM was recorded. Endogenous tryptophan fluorescence measurements, both in the presence and absence of liposomes, demonstrated that toposome tertiary structure was sensitive to increasing concentrations of calcium with an apparent dissociation constant (calcium) of 240 μM. Toposome-driven, liposome aggregation demonstrated a similar calcium-concentration dependence. Interestingly, the secondary structural change was required to facilitate toposome binding to bilayers while the tertiary structural change correlated with toposome-driven, membrane-membrane interaction. -- We also showed that the thermal denaturation profile of toposome is dependent upon calcium. Following a calcium-induced change in secondary structure, toposome was increasingly resistant to thermal denaturation. However, the calcium-induced change in tertiary structure rendered toposome more susceptible to thermal denaturation when compared to toposome following the secondary structural change. We also performed chymotryptic digestions in the presence of varying concentrations of calcium. The calcium-induced, secondary structural change had no effect on the chymotryptic cleavage pattern. Similarly, following the tertiary structural change, the chymotryptic digestion profile again remained unchanged. Interestingly, the chymotryptic digestion pattern of toposome bound to phosphatidylserine liposomes did vary as a function of calcium concentration. -- In an effort to define the nature of the calcium binding sites on toposome, we investigated the interaction of this protein with various metal ions. Calcium, Mg²⁺, Ba²⁺, Cd²⁺, Mn²⁺ and Fe³⁺ all bound to toposome. In addition, Cd²⁺ and Mn²⁺ displaced Ca²⁺, prebound to toposome. -- Also in this study we have expanded the analysis of toposome-membrane interaction by probing for toposome-induced changes to the lipid bilayer. Solid-state ²H-NMR allowed us to define the nature of the association of toposome with the bilayer. We found that this protein interacts peripherally with the membrane and that this interaction is facilitated by the calcium-driven secondary structural change in toposome. In addition, we also examined the toposome-bilayer interaction using atomic force microscopy. In the absence of added calcium little or no binding of toposome occurred. In contrast, the calcium-induced secondary structural change caused significant binding of toposome to the bilayer. Following the calcium-induced tertiary structural change no further binding of protein to the bilayer was observed. -- We utilized immunogold labeling to define the subcellular localization of toposome in the sea urchin egg and embryo. In the unfertilized egg, toposome was found on the entire cell surface as well as stored in two different compartments, the yolk and cortical granules. Mitochondria and lipid vacuoles were not labeled. Label continued to be detected in the yolk granules and on the cell surface throughout development. -- Collectively, these results provide a structural basis for a previously described role for toposome in mediating biologically relevant, membrane-membrane interactions. Calcium was found to induce two calcium concentration-dependent structural transitions in toposome: the first structural change was required to facilitate toposome binding to the membrane, while the second structural change enabled this protein to drive membrane-membrane adhesive interactions.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Includes bibliographical references (leaves 158-177)|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Sea urchins--Eggs; Sea urchins--Embryos|
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