Hsp70 is a conserved molecular chaperone which plays an indispensable role in regulating protein folding,oligomerization,and degradation in prokaryotic and eukaryotic organisms.Hsp70 consists of an N-terminal nucleotide binding domain,a beta-sheet-containing substrate binding domain and an alpha-helical C-terminal lid domain.It is an ATP-dependent chaperone which hydrolyzes ATP to ADP resulting in inter-domain confonnational changes and cycling between substrate-binding and release states.However,the dynamics of the ATP hydrolysis-driven conformational cycle is still not clear.Here,using human Hsp70 as a model,we studied the changes in conformational distribution of Hsp70 under different nucleotide and substrate binding states by single molecule FRET.The results show that the conformation of its apo and ATP states are heterogeneous,while its ADP state is uniform.Although small substrates have no effect on the conformation of Hsp70,larger substrates could change the relative position between substrate binding domain and nucleotide binding domain.We also studied the kinetics of ATP-induced conformational changes in real time by ensemble FRET.We observed a fast process which was related to nucleotide binding followed by a slow process which was assigned as the ATP-induced domain movement.These results provide new insight into the allosteric mechanism of Hsp70.