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| Abstract Title:
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| Analyzing Protein Interactions & Dynamics: Spatial Distribution of Molecular Chaperone/Co-Chaperones Evident at a Sub-organelle Level in Saccharomyces cerevisiae
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| Graduate Student Presenter:
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Carissa L. Young
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| Name of the Author(s) and Affiliation(s):
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Carissa L. Young, David Raden, Anne S. Robinson, Department of Chemical Engineering, University of Delaware; Marc Griesemer, Linda Petzold, Department of Computer Science, UCSB; Francis Doyle III, Department of Chemical Engineering, UCSB
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BiP/Kar2, the molecular chaperone of the endoplasmic reticulum (ER), is identified in multiple critical processes of the cell including ER translocation and protein folding. Biochemical and genetic experiments have demonstrated BiP’s association with selective co-chaperones for specific cellular processes. We hypothesize that chaperone/co-chaperone interactions are a result of spatial inhomogeneity, regulated by co-chaperones, and this inhomogeneity serves to dictate cellular functions. The low resolution of traditional immunofluorescence techniques combined with BiP’s relative high abundance has previously inhibited the determination of molecular gradients. A systems biology framework, including a hypothesis-driving approach and integration of experimental and computational design, will elucidate the mechanisms associated with chaperone/co-chaperone interactions.
Advances in confocal light microscopy (CLM) combined with the use of green fluorescent protein (GFP) allow the continuous monitoring of protein dynamics in living cells. Examining the kinetics of a system requires in vitro techniques that provide a direct measurement of equilibrium and reaction rate constants. Computational models, both deterministic and stochastic, provide a mechanistic-level understanding of these interactions.
Fusion proteins of BiP and co-chaperone Sec63 have established that spatial distribution occurs at a sub-organelle level during translocation; confirmed that the ER of S. cerevisiae is continuous and structurally dynamic; and determined that the ER morphology changes as a result of induced stress. Recombinant protein expression and purification of BiP and Sec63 have been accomplished. Preliminary in vitro studies will determine estimates of kinetic parameters to verify existing computational results. Additional insights provided by computational simulation account for spatial and temporal effects of chaperones/co-chaperones.
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