Olası koşaperon MZB1 proteininin Grp94 şaperonu üzerindeki etkisi

Grp94 is Hsp90 paralog, residing in endoplasmic reticulum (ER), implicated in efficient expression of various proteins secreted and presented on cell membrane. As an essential eukaryotic chaperone, a set of studies have already shown that Grp94 involves in maturation of Toll-like receptors, integrin family members and antibody secretion. Since the condition of ER significantly differs from that of the cytosol, Grp94 must have been uniquely evolved from its cytosolic counterparts in order to deal with distinct client profile of ER. It is known that vast majority of Grp94 clients possess at least one disulphide bond whose formation is crucial to attain 3-D structure and stability for many ER proteins. Besides, ER is much more oxidizing than cytosol due to its enzymatic components catalysing disulphide formation. Despite of having same domain organisation with that of other Hsp90 family members, in which N terminal domain preceding M domain and finally C-terminal domain functioning in dimerization, Grp94 has significant conformational differences correspond to its cytosolic counterparts. Most importantly, unlike other Hsp90, in the presence of AMP-PNP or ADP, Grp94 adopts same twisted V conformation which seems to make N-terminal dimerization unfavourable. It must be noted that binding of ATP makes Hsp90 family members undergo some conformational changes in which N- terminal domains of each protomer dimerize resulting in ATP hydrolysis. Furthermore, unlike its cytosolic counterparts, Grp94 does not show different affinity profile for ATP and ADP. These data has been leading an intriguing question regarding to the extent in which Grp94 differs from its cytosolic homologs in undergoing conformational changes during its ATPase cycle. It is long standing enigma as to whether Grp94 is regulated by a co-chaperone which may mediate client loading or remodel ATPase cycle to function in maturation of a set of ER proteins. Even though some studies have shown that a set of protein interact Grp94 along with clues concerning physiological relevance, our current understanding of the precise nature of interaction in these presumptive proteins with Grp94 and their regulatory effects on Grp94 conformational changes are still infant. pERp1/MZB1 is relatively newly discovered protein residing in ER. Its function has not been fully understood, which presumably act as unique oxidoreductase or chaperone. In previous studies, it has been demonstrated that pERp1 interacts with ER chaperones, Grp94 and BiP. In the context of this thesis, wild type Grp94 and a flanked at both terminal domains by partial ubiquitin version of Grp94 are compared by subjecting to ATPase assay and circular dichroism spectroscopy. The main aim is to check whether ubiquitin tagged Grp94 has no significant difference from wild type in ATPase kinetics and secondary structure. It must be noted that this version was important to do further investigation on Grp94 by single molecule study. Moreover, titration of pERp1 has been performed to calculate EC50 which is an indicator of binding affinity. Apart from steady state kinetics, citrate synthase aggregation assay and analytical size exclusion chromatography are applied to the respective proteins.