The small heat shock proteins are the ubiquitous proteins found in a wide range of organisms and function as molecular chaperones by binding to the folding intermediates of their substrates. Although the crystal structure of HSP16.5, a small heat shock protein from Methanococcus jannaschii, revealed that it is a hollow sphere composed of 24 identical subunits, its activation mechanism remains unclear. We found out that HSP16.5 is active only at high temperatures and forms a stable complex with substrate in a stoichiometric manner. We also observed that the conformational change of HSP16.5 is correlated with the increasing hydrophobic site and its activation as a molecular chaperone. However, it is revealed that the conformational change is not accompanied with the change of the secondary structure of a subunit, but correlated with the increasing diameter of HSP16.5. Therefore, it is proposed that the activation mechanism of HSP16.5 involves temperature induced conformational change with size increment of the complex resulting in the exposure of hydrophobic substrate-binding site. (C) 2003 Elsevier Inc. All rights reserved.