Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce compleme nting defense mechanisms. Organisms adapt to high external salinity by accu mulating small organic compounds known as osmolytes, which equilibrate cell ular osmotic pressure. Osmolytes can also act as "chemical chaperones" by i ncreasing the stability of native proteins and assisting refolding of unfol ded polypeptides. Adaptation to heat stress depends on the expression of he at-shock proteins, many of which are molecular chaperones, that prevent pro tein aggregation, disassemble protein aggregates, and assist protein refold ing. We show here that Escherichia coli cells preadapted to high salinity c ontain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which esc aped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chape rone activity in vitro. Systematic dose responses of glycine betaine, glyce rol, proline, and trehalose revealed a regulatory effect on the folding act ivities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proli ne, glycerol, and especially glycine betaine activated the molecular chaper ones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentr ations, especially trehalose, strongly inhibited DnaK-dependent chaperone n etworks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity af fects dynamic interactions between chaperones and folding substrates and st abilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated dis aggregation and refolding by modulating the intracellular levels of differe nt osmolytes.