ABSTRACT
Data of this study showed that alphaD-alphaE helices and the conserved interdomain linker are two interfaces essential not only for the self-association but also for the functional properties of rat HSC70. Self-association which is a conserved property of HSP70 seems to be important for the activity of these proteins.
Subject(s)
HSC70 Heat-Shock Proteins/genetics , Adenosine Triphosphatases/metabolism , Animals , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , HSC70 Heat-Shock Proteins/metabolism , Protein Structure, Quaternary , Rats , Sequence Deletion , UltracentrifugationABSTRACT
The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His(+)/C30WT and C30DeltaL-His(+)/C30DeltaL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30DeltaL-His(+) but has no effect on the elution profile of C30WT-His(+), compared to their respective untagged forms C30DeltaL and C30WT. These observations were also confirmed by AU analysis which indicates that C30DeltaL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.
Subject(s)
HSP70 Heat-Shock Proteins/chemistry , Histidine/chemistry , Oligopeptides/chemistry , Animals , Chromatography, Affinity/methods , Chromatography, Gel/methods , Dimerization , Electrophoresis, Polyacrylamide Gel , HSP70 Heat-Shock Proteins/analysis , HSP70 Heat-Shock Proteins/genetics , Models, Molecular , Mutant Proteins/analysis , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation/genetics , Rats , Recombinant Proteins/analysis , Recombinant Proteins/chemistryABSTRACT
Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.
