Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

Abstract

In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (T_m). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of T_m measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of T_m variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of T_m for 58 of 61 Plasmodium proteins tested. However, with several proteins, T_m could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in T_m measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. (Journal of Biomolecular Screening 2009:700-707)