Coordination between N- and C-terminal Kinetics of Hsp90 Investigated by SmFRET

Abstract

Hsp90 is a molecular chaperone required for the activation of a large amount of client proteins and survival of the cell during heat shock. It consists of two monomeric chains with dimerization interfaces at the C and N-terminal end[1]. Its chaperone function is dependent on ATP binding and hydrolysis as well as N- terminal and C-terminal dimerization[2]. Up to now mainly the N-terminal dimerization kinetics of Hsp90 has been investigated[3], while the C-terminal interface was assumed to be closed for many minutes, because of its low equilibrium binding constant. We developed a fluorescent based single molecule assay, which allows to investigate C-terminal dimerization independent of N-terminal kinetics. Surprisingly, we find C-terminal dissociation / association kinetics on the timescale of seconds. In addition, this kinetics is nucleotide dependent although the nucleotide binding pocket is far away in the N-terminal domain. Therefore, we conclude that there is coordination through the complete Hsp90 monomer. These findings are confirmed by well defined N-terminal mutations[4]. [1] Ali M.M. et al. Nature (2006) 440, 1013-1017. [2] Wegele H., L. Müller, J. Buchner; Rev Physiol Biochem Pharmacol (2004) 151, 1-44. [3] Mickler M., M. Hessling, C. Ratzke, J. Buchner, T. Hugel, NSMB, 16, 281 (2009) [4] Ratzke C., M. Mickler, J. Buchner, T. Hugel, Manuscript in prep.