hsp70-protein complexes. Complex stability and conformation of bound substrate protein.

The presence of bound substrate protein increases the thermostability of hsp70 molecular chaperones (heat shock proteins of molecular mass 70 kDa). Complexes between hsp70 and unfolded substrate proteins were isolated by size-exclusion high performance liquid chromatography. The isolated complexes were observed to dissociate at a significant rate even in the absence of ATP. The presence of ADP caused a substantial increase in the stability of the complex. Both ADP and inorganic phosphate were found to inhibit the ATP-induced dissociation of complex. ADP was also observed to increase both the rate of complex formation and its stability as a function of temperature, suggesting an important regulatory role for nucleotides during heat shock. Circular dichroism and fluorescence studies of the complex between DnaK and a thermally unstable mutant of staphylococcal nuclease indicate that the bound substrate protein is significantly unfolded. A model for hsp70 cycle of complex formation and dissociation, which accounts for the regulatory role of nucleotides, is proposed.