Analyses of the chaperone activities of HspA His

Citrate synthase (CS) and insulin have often been used as model substrates to study the influence of small heat shock proteins on protein folding and unfolding reactions (Farahbakhsh et al., 1995; Jakob et al., 1993).

2.7.6.1. The interaction of HspAHis with chemically denatured CS

To test whether HspA_His can interact with an unfolded protein and facilitate protein folding, the effect of HspA_His on folding of CS was studied. The mitochondrial CS, from pig heart (Boehringer, Mannheim), is a homodimer with a molecular mass of 49 kDa.

15 µM CS (dimer) was denatured in 6 M guanidine hydrochloride (GuHCl) buffer at 25 °C for two hours. After a 1:100 dilution in 40 mM HEPES (pH 7.5) buffer, the refolding procedure of denatured CS was initiated (Fig. 2.32). When chaperone is absent in the dilution buffer, CS aggregates immediately as shown by an increase of light scattering. Aggregation is due to an incorrect folding.

0.075 µM HspA 0.015 µM HspA 0.003 µM HspA 0.15 µM IgG Control

0 10 20 30 40

time (min)

0 10 20 30

relative light scattering

Fig. 2.32. Aggregation of chemically denatured CS in the absence and presence of HspA_His. The kinetics of aggregation was determined by light scattering. Guanidine hydrochloride(GuHCl) denatured CS was diluted to a final concentration of 0.03 µM (monomer). The concentrations of HspAHis and bovine IgG are indicated in the graph.

The presence of HspAHis in the dilution buffer suppressed spontaneous aggregation of CS.

Increasing amounts of HspAHis reduced the degree of CS aggregation. The addition of 0.015 µM HspAHis (oligomer) in the dilution buffer suppressed the spontaneous aggregation of CS almost completely. Bovine IgG was used as a control protein in this assay, which had no effect on the refolding of CS. Two known small heat shock proteins were tested also in this aggregation assay. One is α-crystallin from bovine eyes, and the other is Hsp25 from mouse (kindly provided by M. Ehrnsperger, University of Regensburg, Germany). Both proteins did not prevent the aggregation of the chemically denatured CS (data not shown).

2.7.6.2. The influence of HspA_His on the chemically induced aggregation of insulin

Insulin was selected to investigate whether HspA_His can prevent aggregation during its unfolding. Insulin, from bovine pancreas (Sigma), is composed of A-chain and B-chain that are joined by two interchain disulphide bonds. The A-chain contains an extra intrachain disulphide bond. The molecular mass of insulin is about 5.8 kDa, and that of the B-chain is 3.3 kDa. Reduction of the insulin interchain disulphide bonds leads to aggregation and precipitation of the B chain while the A chain remains in solution (Farahbakhsh et al., 1995).

Fig. 2.33 shows that the B-chain of insulin underwent spontaneous aggregation after addition of dithiothreitol (DTT) to a final concentration of 20 mM. When mouse Hsp25 was present in the buffer before the addition of DTT, the spontaneous aggregation of the B-chain was suppressed. However, HspA_His did not prevent the aggregation of the B chain of insulin.

0 20 40 60 80

0 5 10 15 20

time (min)

Control HspA 0.15 µM Hsp25 0.15 µM

relative light scattering

Fig. 2.33. Aggregation of the B chain of insulin after addition of dithiothreitol (DTT) to a final concentration of 20 mM in the absence or presence of HspAHis or Hsp25. The kinetics of aggregation was determined by measuring light scattering of the samples. Insulin was diluted to a final concentration of 0.25 mg/ml. The concentrations of HspAHis and Hsp25 used are indicated in the graph.

2.7.6.3. The effect of HspA on the reactivation of chemically denatured CS

To understand how HspA_His interacts with denatured CS, the recovery of the enzymatic activity of unfolded CS was determined.

Under the chosen conditions, when chemically denatured CS was diluted in the dilution buffer, the specific activity of CS recovered spontaneously up to about 20% of that of native CS in the absence of chaperone. Surprisingly, in the presence of HspAHis in the dilution buffer, spontaneous reactivation of CS was completely blocked (Fig. 2.34). This may be due to a stable complex formed between unfolded CS and HspA_His. Bovine IgG did not affect spontaneous reactivation of unfolded CS (data not shown).

The chaperone function of small heat shock proteins needs a cooperation with a cofactor or other chaperones (Ehrnsperger et al., 1997; Veinger et al., 1998). Therefore, it was tested whether such factors might help to dissociate the HspAHis-CS complex. It had been reported that Hsp70 or oxaloacetic acid (OAA), a substrate and ligand of CS, could trigger the dissociation of the complex formed by the thermally denatured CS and mouse Hsp25. The same approach was used to test whether these factors might facilitate the reactivation of HspA_His bound CS.

0 5 10 15 20

2 12 22

time (min)

0.15 µM HspA Control

reactivation (%)

0.15 µM HspA + 1 mM OAA 0.15 µM HspA + 3mM ATP 0.15 µM HspA + 0.6 µM Hsp70 + 3mM ATP

Fig. 2.34. Reactivation of unfolded CS in the absence and presence of HspAHis. Unfolded CS was diluted in a buffer without HspA_His or with 0.15 µM HspA_His to a final concentration of 0.15 µM. 30 minutes after dilution of denatured CS, Hsp70 or OAA or ATP was added to the reaction, individually or simultaneously as indicated in the graph.

After incubation of HspAHis with unfolded CS for 30 minutes, Hsp70 from yeast (kindly provided by M. Ehrnsperger, University of Regensburg, Germany) or OAA was added to the dilution buffer. As shown in Fig. 2.34, neither Hsp70 nor OAA led to the dissociation of bound CS from HspA_His. As measured by its activation, CS activity detected was slightly higher than zero. This indicates that ATP had no effect on stimulation of CS reactivation in cooperation with Hsp70.