Preheat treatment for Mycobacterium tuberculosis Hsp16.3: Correlation between a structural phase change at 60 degrees C and a dramatic increase in chaperone-like activity

The in vitro chaperone-like activity of Mycobacterium tuberculosis small he at shock protein Hsp16.3 was found to be dramatically enhanced to the same extent after preheat treatment at or over 60 degreesC. Structural analysis using gel filtration, native pore-gradient PAGE, nondenaturing PAGE, and fa r-UV CD spectroscopy consistently revealed no significant difference betwee n the native and the preheated Hsp16.3 proteins. However, near-UV CD spectr oscopy clearly demonstrated that the tertiary structure of preheated Hsp16. 3 is quite similar to its native conformation, with a minor but significant difference. Further analysis using differential scanning calorimetry indic ated that Hsp16.3 exhibited a structural transition near 60 degreesC. Ah th ese results together indicate that Hsp16.3 suffers a phase change at approx imately 60 degreesC, which seem to remove a structural energy barrier for t he protein to refold to a conformational status with increased chaperone-li ke activity, (C) 2001 Academic Press.