DnaK from Thermus thermophilus (TDnaK) is unique because significant fractions of cellular TDnaK exist as a trigonal K.J complex that consists of three copies each of TDnaK, TDnaJ, and an assembly factor TDafA. Here, chaperone functions of the K.J complex and free TDnaK plus free TDnaJ (K+J) were compared. Substrate proteins were completely denatured at 72-73 degrees C or 89 degrees C in the absence or the presence of K.J complex or K+J and were subsequently incubated at a moderate temperature of 55 degrees C. TGrpE and ATP were always included in the K.J complex and K+J, and TClpB was supplemented at 55 degrees C. At 72-73 degrees C, both the K.J complex and K+J suppressed heat aggregation of substrate proteins. During the next incubation at 55 degrees C, K+J, assisted by TClpB, was able to disaggregate the heat aggregates and efficiently reactivate activities of the proteins, whereas the K.J complex was not; it reactivated only the soluble inactivated proteins. When substrate proteins were heated to 89 degrees C, both the K.J complex and K+J were no longer able to prevent heat aggregation, and because of selective, irreversible denaturation of TDafA the K.J complex dissociated into K+J, which then exhibited disaggregation activity during the next incubation at 55 degrees C. Thus, TClpB-assisted disaggregation activity belongs only to K+J, and TDafA is a potential thermosensor for converting the K.J complex to K+J in response to heat stress.