The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

Abstract

The heat shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is an organism that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5 and the intracellular pH can be as low as 5.0. This study was performed to test the ability of Hsp60 from H. pylori to function as a molecular chaperone under mildly acidic conditions. We report here that Hsp60 could suppress the acid-induced aggregation of alcohol dehydrogenase (ADH) in the 7.0-5.0 pH range. Hsp60 was found to undergo a conformational change within this pH range. It was also found that exposure of hydrophobic surfaces of Hsp60 is significant and that their exposure is increased under acidic conditions. Although, alcohol dehydrogenase does not contain exposed hydrophobic surfaces, we found that their exposure is triggered at low pH. Our results demonstrate that Hsp60 from H. pylori can function as a molecular chaperone under acidic conditions and that the interaction between Hsp60 and other proteins may be mediated by hydrophobic interactions.

Keywords: Acid stress; Conformational changes; Hsp60; Molecular chaperone; Protein aggregation.

Fig. 1

Aggregation of ADH and Hsp60 monitored by using light scattering. ADH (1 μM) or Hsp60 (1 μM) was incubated in 50 mM Tris-HCl (pH 7.5-6.5) or 50 mM sodium citrate (pH 6.0-5.0) at 25 °C. The figure shows the % aggregation of ADH (diagonal bars) and Hsp60 (open bars) relative to the maximal aggregation (100%) corresponding to the highest scattering signal detected for ADH (pH 5.5).

Fig. 2

Time course of the light scattering of ADH. ADH (1 μM) was added after 5 min to 50 mM sodium citrate, pH 5.5 at 25 °C without (closed circles) or with 1 μM Hsp60 (open circles).

Fig. 3

Suppression of aggregation of ADH by Hsp60 monitored by using light scattering. Hsp60 (1 μM) was incubated in 50 mM Tris-HCl (pH 7.5-6.5) or 50 mM sodium citrate (pH 6.0-5.0) at 25 °C. ADH (1 μM) was added after 5 min and light scattering was recorded for 30 min after the addition of ADH to the buffers.

Fig. 4

Tryptophan fluorescence of the Y359W mutant of Hsp60. The Y359W mutant was incubated with 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate pH 5.5 and fluorescence recorded as described in “Materials and methods”. The final concentration of the Hsp60 mutant was 2.0 μM (monomers).

Fig. 5

Measurement of exposure of hydrophobic surfaces of Hsp60. Fluorescence emission spectra were recorded for bisANS alone in 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate, pH 5.5 (lowest spectra). Hsp60 was added to bisANS in 50 mM Tris-HCl, pH 7.5 (middle spectrum) or to bisANS in 50 mM sodium citrate, pH 5.5 (highest spectrum). The final concentrations of bisANS and Hsp60 were 10 μM and 1 μM (monomers), respectively.

Fig. 6

Measurement of exposure of hydrophobic surfaces of ADH. Fluorescence emission spectra were recorded for bisANS alone in 50 mM Tris-HCl, pH 7.5 or 50 mM sodium citrate, pH 5.5 (lowest spectra). ADH was added to bisANS in 50 mM Tris-HCl, pH 7.5 (middle spectrum) or to bisANS in 50 mM sodium citrate, pH 5.5 (highest spectrum). The final concentrations of bisANS and Hsp60 were 10 μM and 1 μM (monomers), respectively.