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dc.contributor.advisorWilbanks, Sigurd
dc.contributor.authorCampbell, Eugene
dc.date.available2018-11-04T20:52:59Z
dc.date.copyright2018
dc.identifier.citationCampbell, E. (2018). Comparison of FRET probes of conformational changes in DnaK and Hsc70 (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/8526en
dc.identifier.urihttp://hdl.handle.net/10523/8526
dc.description.abstractHsp70s are one of the most well conserved protein families known, yet little of their in vivo activity is understood and experimental resources required to investigate their activity. All Hsp70s are made up of two highly conserved functional domains joined by a flexible linker, the nucleotide binding domain and the substrate binding domain. The Hsp70 proteins undergo a conformational in their peptide binding and release cycle, with conformation of the protein determined by the nucleotide bound in the nucleotide binding domain. This study compared the two most wildly studied Hsp70 proteins, DnaK and the human Hsc70, HSPA8. Hsc70 is a constitutively expressed member of the Hsp70 family which has many roles within the cell, assisting with folding of nascent polypeptides, targeting proteins for degradation and translocating proteins to the endoplasmic reticulum. Both proteins were purified from bacterial expression systems and the purification of Hsc70 was improved by the addition of an additional isocratic step during ion exchange chromatography. Efficiency of labelling with fluorescent dyes was improved and the activity cycle of DnaK and Hsc70 directly compared. Of the eight variants tested only two, DnaK variant C16S/K321C/E430C and Hsc70 variant E318C/T427C/C574S/C603S showed changes in FRET efficiency in response to different nucleotides. In order to validate these variants as suitable models and to probe why other variants showed no changes in FRET efficiency, proteins were analysed for their protein refolding activity. An improved luciferase refolding assay was developed to analyse the rate of protein refolding for each of the Hsc70 and DnaK variants. All of the variants for both DnaK and Hsc70 retained protein refolding activity, however most were less efficient than the wild type protein, showing between 20 to 100% of the wild type proteins activity. This study identified Hsc70 variant E318C/T427C/C574S/C603S as a suitable candidate for future in vivo fluorescence assay of Hsc70 function.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectProtein
dc.subjectdynamics
dc.titleComparison of FRET probes of conformational changes in DnaK and Hsc70
dc.typeThesis
dc.date.updated2018-11-04T07:28:01Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.openaccessOpen
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