Regulation and Mechanism of Action of the Small Heat Shock Protein from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105°C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105°C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56°C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50°C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.

Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosus

“…Our results indicated that Pfds and sHsps have analogous roles as they both improve the efficiency of Hsp60 catalysis. Hsp60 and Pfd subunits in P. furiosus are constitutively expressed (data not shown) whereas the sHsp is highly induced by heat shock treatment at 1058C (Laksanalamai et al, 2001). In addition, after P. furiosus cells are removed from heat shock conditions and restored to growth conditions (958C), the levels of mRNA and protein appear to decrease rapidly as detected by Northern and Western blot, respectively (Laksanalamai, Lowe and Robb, unpublished results).…”

“…Pyrococcus furiosus sHsp and Hsp60 were cloned, expressed, and purified as previously described by Laksanalamai et al (2001) and Emmerhoff et al (1998), respectively. The genes encoding P. furiosus Pfd a and b subunits were identified based on the homology between the P. furiosus and P. horikoshii sequences (Okochi et al, 2002).…”

“…A stock solution of Taq polymerase (5U/mL, Cat: FB600030 Lot: 005975 ) was diluted five-and tenfold to provide final concentrations of 0.025, 0.005, and 0.0025 U/mL in the PCR reactions. From a stock solution of a purified recombinant sHsp in Buffer A (25 mM potassium phosphate buffer, pH 7.0, 2 mM dithiothreitol, and 1 mM EDTA) (Laksanalamai et al, 2001), sHsp was added to the PCR reaction providing a final concentration of 0.2 mg/mL. Control reactions without sHsp were performed with the addition of buffer A and water to adjust the total volume to 50 mL.…”

“…We reported previously that the sHsp from Pyrococcus furiosus (Pfu-sHsp) can prevent bovine glutamate dehydrogenase from aggregating at 508C (Laksanalamai et al, 2001). During PCR reactions, the temperature fluctuates over a wide range, in this case, 50-958C.…”

“…The most extensively studied chaperone in P. furiosus is the sHsp, which is an a-crystallin homolog with conserved sequence motifs in common with sHsps and crystallins from all domains of life (Chang et al, 1996;Haley et al, 2000;Kim et al, 1998;Laksanalamai et al, 2001Laksanalamai et al, , 2003van Montfort et al, 2001). Several lines of evidence indicate that sHsps can prevent denatured proteins from aggregating but are unable to refold non-native proteins in a catalytic fashion (Chang et al, 1996;Laksanalamai et al, 2001). Hsp60s on the other hand catalyze ATP-dependent protein folding (Hartl, 1996;Hartl and Hayer-Hartl, 2002).…”