Generating recombinant citrate synthase as substrate to study the function of molecular chaperones

Citrate synthase is a well-established substrate to measure the relative activity of molecular chaperones at low concentrations and with high precision. Under denaturing conditions, citrate synthase unfolds and forms monomeric intermediates prone to aggregate at elevated temperatures. Aggregation can be monitored by measuring an increase in light scattering. Chaperone binding will prevent its aggregation and mitigate light scattering depending on the molar excess of a chaperone added over citrate synthase. Here, we present a convenient and cost-efficient method to produce porcine citrate synthase from E. coli to circumvent the need for commercial citrate synthase purified from pig hearts. The recombinant citrate synthase is catalytically active and functions upon chemical denaturation as a substrate in the aggregation assay, aggregating with similar kinetics as commercial citrate synthase and resulting in reproducible measurements for the redox-regulated chaperone function of Get3. Aggregation strongly depends on substrate concentration, temperature and pH, whereas the addition of DTT or low amounts of salt barely affected light scattering. We further applied the citrate synthase aggregation assay to determine and compare relative chaperone activities of Get3 induced by hydrogen peroxide in its nucleotide-bound and -free conformation and showed that hypochlorous acid readily activated Get3's chaperone function independent of its nucleotide-binding state. Generating large amounts of recombinant citrate synthase which is stable over long time and can be used as a substrate in aggregation assays will facilitate thorough in vitro analyses of molecular chaperones, their activation mechanisms and functional regulation.