Abstract
The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and NdeI. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and NdeI) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a Mycobacterium tuberculosis protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/NdeI aggregation. Figure 1.Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I1, I2) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.