Chemical Chaperones Regulate Molecular Chaperones in Vitro and in Cells under Combined Salt and Heat Stresses

Abstract

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK؉GroEL and DnaK؉ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Proline to the rescue

“…For example, osmolyte-enhanced folding͞anti-aggregation could be further augmented by the simultaneous increase in select molecular chaperones, particularly those involved in folding. From a biotechnology prospective, using in vitro and in vivo osmolyte͞chaperone protein combinations could also result in a dramatic increase in the levels of correctly folded proteins (16)(17)(18). Alternatively, one could examine the possibility that combinations of osmolytes may also facilitate protein folding.…”

Soluble Expression of Recombinant Human Smp30 for Detecting Serum Smp30 Antibody Levels in Hepatocellular Carcinoma Patients

“…Betaine is an osmosis-regulator; the presence of physiological amounts of osmosis-regulator can increase osmolyte viscosity and reduce the thermodynamically less favorable accidental misfolding events, so significantly increase the stability of native thermolabile proteins (Diamant et al, 2001;Timasheff et al, 2002). In our experiments, pTf16 co-expression system added betaine and benzyl alcohol could promote the expression level of soluble SMP30 more effectively, which means adding adjuvant is an effective method for soluble expression.…”

Structural Genomics of Minimal Organisms: Pipeline and Results

“…Simple heat shock before induction is known to enhance the solubility of some recombinant proteins produced in E. coli (Chen et al, 2002). Some osmolytes behave as "chemical chaperones" by promoting the correct folding of unfolded proteins in vitro and in the cell (Samuel et al, 2000;Yang et al, 1999;Voziyan and Fisher, 2000;Diamant et al, 2001). We have combined these two elements, heat shock and high salt media, to increase the fraction of soluble protein produced from targets.…”