The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: Conformational flexibility and molecular chaperone activity

Hsp 16.3, the alpha-crystallin-related small heat shock protein of Mycobact erium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer- of-trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J Biol Chem 271:7218-7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates w ith dramatically increased chaperone activities were detected after treatme nt with very low concentrations of guanidine hydrochloride (0.05 M). urea ( 0.3 M), or mild heating (30 degrees C). The intermediates showed a signific ant increase in their capacity to bind the hydrophobic probe 1-anilino-8-na phthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 degree s C. CD spectroscopy studies revealed no significant changes in protein sec ondary and tertiary structures at these mild treatments. Our in vitro studi es also indicate that long-time-heated Hsp16.3, heated even to temperatures as high as 85 degrees C, has almost the same, if not a slightly greater, c haperone activities as the native protein when cooled to room temperature a nd its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophob ic surfaces and that the protein structure is highly stable and flexible, t hus highly adapted for its function.