ABSTRACT
In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB
Tha
) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB
Tha
forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB
Tha
reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE
Tha
) and ATP. Interestingly, the mixture of dimer and monomer ClpB
Tha
, which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB
Tha
forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE
Tha
and ATP under poststress conditions.
Analysis of serum lipoproteins by high performance gel filtration chromatography.
