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 (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential
ligands. The authors present an investigation of Tm 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 Tm for 58 of 61 Plasmodium
proteins tested. However, with several
proteins, Tm 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 Tm measurements increases the success of thermal melt screens involving
proteins for which a standard
buffer is suboptimal.