[27] Analysis of chaperone function using citrate synthase as nonnative substrate protein

The chapter presents a study related to the analysis of chaperone function by using citrate synthase (CS) as nonnative substrate protein. The chapter describes the use of CS as a chaperone substrate. CS is well suited, because unfolding and refolding can be easily monitored and both the thermal unfolding, as well as the folding pathway of chemically denatured CS is known in some detail. The influence of the four major classes of molecular chaperones—small heat shock proteins (Hsps), GroEL, Hsp70, and Hsp90—on CS folding has been characterized. The use of CS as a substrate protein for molecular chaperones allows one to address whether chaperones suppress aggregation, undergo stable or transient interactions with the substrate protein, interact with early- or late-unfolding intermediates, act in an adenosine triphosphate (ATP) -dependent manner, and resemble a member of one of the major classes of molecular chaperones. Citrate synthase (EC 4.1.3.7) is a commercially available, dimeric, mitochondrial protein, composed of two identical subunits (48.969 kDa each). The thermal unfolding of CS can be induced in vitro at temperatures similar to heat shock temperatures in vivo. Depending on the functional mechanism of the individual chaperone, aggregation can be affected differently.