Within the field of biochemistry, chaperone and co-chaperone proteins are of important significance due to their vital role in assisting protein folding. ERdj6 is an Hsp40 co-chaperone that works with Hsp70 to facilitate client binding and folding. ERdj6 was grown and purified in E. Coli, with wild-type and mutant protein samples grown. Within Hsp40 proteins there is a highly conserved HPD tripeptide, we wanted to determine whether this tripeptide was necessary for activity. Mutagenesis was performed in order to convert the HPD motif into a QPD motif. The WT sample’s activity with the Hsp70 chaperone BiP was tested in an ATPase assay, the QPD mutant underwent similar testing and the final activities were compared. The QPD mutant was found to have significantly lower activity than the WT protein. A sample of the WT protein where the polyhistidine tag (used for affinity chromatography and purification) had been cleaved, was also tested using these ATPase assays and the activity of the cleaved protein was found to be essentially the same as samples that still contained the polyhistidine tag. The oligomeric structure of ERdj6 was also of interest and native PAGE analysis was performed on the QPD, uncleaved WT, and cleaved WT samples to determine if an oligomer was present in solution. These native PAGE analyses showed that all three protein samples were present in both a trimeric and monomeric form, and further activity assays are necessary to determine the active form of ERdj6. Oligomer databases were also used to predict the trimeric structure of ERdj6 and mutations were submitted to the GalaxyWeb Homomer system to predict which residues may be important for oligomerization. This database predicted that the HPD tripeptide and the residues surrounding it are vital for trimerization. Further study into this region and ERdj6’s oligomerization are necessary.
Author: AJ Morton
Advisor: Andrea Kravats, Chemistry and Biochemistry
Graduate Advisor: Erin Unruh, Chemistry and Biochemistry
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