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Abstract
The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.
Highlights
The chaperonin GroEL from Escherichia coli belongs to a class of proteins, termed molecular chaperones, whose collective function is to assist in the folding of newly synthesized proteins and in the refolding of non-native polypeptides generated under conditions of stress
Increased GroEL ATPase Activity at High Concentrations of Ammonium Ions—A key feature of the GroEL mechanism of action is its ATPase activity, which is modulated by interaction of GroES and substrate protein with the chaperonin [4, 7, 8, 40, 41]
We investigated the effect of high salt concentrations on GroEL ATPase activity
Summary
Proteins—GroEL, and GroES, were expressed in and purified from E. coli as described previously [8, 17]. Analysis of GroEL Stability—GroEL (530 nM) was diluted at 75 °C into buffer containing 25 mM MOPS-NaOH, pH 7.6, 5 mM MgCl2, and increasing concentrations of (NH4)2SO4 as indicated in the figure legends. For spontaneous refolding, denatured GFP was diluted 200-fold into 1 ml of buffer B (25 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, 5 mM dithiothreitol) supplemented with either 50 mM KCl or 1.2 M (NH4)2SO4. To determine the maximum fluorescence intensity of a given amount of bisANS that is totally bound by protein, 250 nM of bisANS in KCl- or (NH4)2SO4-containing buffer was titrated with increasing amounts of GroEL and graphical analysis of the resultant fluorescence emission spectra was carried out according to Bohnert et al [39]. The excitation and emission slit widths were set at 5 and 3 nm of bandpass, respectively
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