Abstract

Many diseases in humans are caused by mutations that decrease the stability of specific proteins or increase their susceptibility to aggregation. Consequently, the availability of high-throughput methods for assessing protein stability and aggregation properties under physiological conditions (e.g., 37 degrees C) is necessary to analyze physicochemical properties under conditions that are closer to in vivo models. Therefore, the authors have explored the use of isothermal denaturation (ITD) in a 384-well format to evaluate the reproducibility of the method in assessing the stability of proteins at temperatures below the melting temperature and detecting the binding of ligands. Under the conditions tested, the authors were able to assess the stability of citrate synthase and malate dehydrogenase at different constant temperatures and detect the binding of oxaloacetate and nicotinamide adenine dinucleotide to these 2 enzymes, respectively, using the 384-well format. The ITD experiments detected ligand binding to these proteins at about 4 times lower concentration compared with techniques that measure changes in melting temperature. The data show that ITD can be applied to screen libraries of a relatively large number of compounds or detect small stability differences between protein variants.