ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins. The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions. Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. We expressed and purified the N-terminal fragment of ClpB (residues 1-147). Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure. Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain. The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN). This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN. Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein. As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity. We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase. Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates. Study holds ProTherm entries: 14875 Extra Details: 10% glycerol and 1mM DTT was added in the experiment,amino-terminal domain ClpB; molecular chaperone; folding domain; protein stability; protein-protein,interactions; differential scanning calorimetry
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||2|
|Proteins||Chaperone protein ClpB ; Chaperone protein ClpB|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence HMQALKKYTIDLTERAEQGKLDPVIGRDEEIRRTIQVLQRRTKNNPVLIGEPGVGKTAIVEGLAQRIINGEVPEGLKGRRVLALDMGALVAGAKYRGEFEERLKGVLNDLAKQEGNVILFIDELHTMVGAGKADGAMDAGNMLKPALARGELHCVGATTLDEYRQYIEKDAALERRFQKVFVAEPSVEDTIAILR|