The alteration in the chaperone function of M. leprae HSP18 induced by ATP is reversible in nature.
(A) DTT-induced aggregation of 0.05 mg/ml lysozyme (Lys) in the presence/absence of different HSP18 solutions. First, HSP18 was incubated in absence (solution 1) and presence of 1 mM ATP (solution 2) at 25°C for 1 hr. Then, part of solution 1 and 2 were dialysed overnight against 50 mM phosphate buffer, pH 7.5. The resultant solution achieved from the dialysis of solution 1 and 2 were termed as solution 3 and 4, respectively. Subsequently, assay was performed with these solutions. Trace 1: Lys alone; Trace 2: Lys + solution 1; Trace 3: Lys + solution 3; Trace 4: Lys + solution 2; Trace 5: Lys + solution 4. Lysozyme aggregation was initiated by adding DTT and scattering was measured while incubation of assay mixture at 37°C. Panel B represents the percent protection ability of different HSP18 solutions against lysozyme aggregation. The chaperone: client protein ratio used for this assay was 1:1 (w/w). Data are the means ± standard deviation from triplicate measurements. NS = Not significant and **p< 0.005.