Characterization of nucleotide-induced changes on the quaternary structure of human 70 kDa heat shock protein Hsp70.1 by analytical ultracentrifugation.

Hsp70s assist in the process of protein folding through nucleotide-controlled cycles of substrate binding and release by alternating from an ATP-bound state in which the affinity for substrate is low to an ADP-bound state in which the affinity for substrate is high. It has been long recognized that the two-domain structure of Hsp70 is critical for these regulated interactions. Therefore, it is important to obtain information about conformational changes in the relative positions of Hsp70 domains caused by nucleotide binding. In this study, analytical ultracentrifugation and dynamic light scattering were used to evaluate the effect of ADP and ATP binding on the conformation of the human stress-induced Hsp70.1 protein. The results of these experiments showed that ATP had a larger effect on the conformation of Hsp70 than ADP. In agreement with previous biochemical experiments, our results suggest that conformational changes caused by nucleotide binding are a consequence of the movement in position of both nucleotide- and substrate-binding domains.