A Probe for Conformational Change of Hsc70 in Mammalian Cells under Stress Conditions
Hsc70 is a constitutively expressed member of the heat shock protein family. It has an important role in proteostasis and can stabilise nonnative proteins before being further matured in refolding, disaggregation, and degradation processes. Hsc70 switches between ATP and ADP bound states, and the hydrolysis of ATP drives the reaction for folding of the unfolded peptide. Förster resonance energy transfer (FRET) was used to report this conformational shift.Creation of the FRET probe used variant 3T of Hsc70, which has three cysteines. One is buried in the conformation, and two are surface exposed on different domains. Maleimide Alexa Fluor (AF) dyes were applied to make covalent additions to the reduced cysteines, and conformational change was tracked by FRET. A luciferase refolding assay showed that although these mutations do not interfere with conformational change, they hinder refolding capabilities with substrate peptides.Double labelled Hsc70 3T was transduced into HEK293 cells with varying conditions. It was found that unfiltered samples, imaged with Opti-MEM in the wells, and at the suggested PULSin concentration by the manufacturer was enough to detect fluorescence of AF488 and AF594. Heat shock can be used to determine how these conformations differ under stress and has been observed in live cell microscopy. This makes FRET a suitable technique to study conformational change of Hsc70 3T under cellular stress. All steps to establish a method to study FRET in live cells were optimised and could be done if a confocal microscope were available.
Advisor: Wilbanks, Sigurd
Degree Name: Master of Science
Degree Discipline: Department of Biochemistry
Publisher: University of Otago
Keywords: New Zealand; Hsc70; FRET; HEK293; CellularStress
Research Type: Thesis