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

Department of Medicine, University of Washington, Seattle, crowther{at}u.washington.edu

Alberto J. Napuli

Department of Biochemistry, University of Washington, Seattle

Andrew P. Thomas

Department of Medicine, University of Washington, Seattle

Diana J. Chung

Department of Medicine, University of Washington, Seattle

Kuzma V. Kovzun

Department of Medicine, University of Washington, Seattle

David J. Leibly

Department of Biochemistry, University of Washington, Seattle

Lisa J. Castaneda

Department of Biochemistry, University of Washington, Seattle

Janhavi Bhandari

Department of Biochemistry, University of Washington, Seattle

Christopher J. Damman

Department of Medicine, University of Washington, Seattle

Raymond Hui

Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

Wim G.J. Hol

Department of Biochemistry, University of Washington, Seattle

Frederick S. Buckner

Department of Medicine, University of Washington, Seattle

Christophe L.M.J. Verlinde

Department of Biochemistry, University of Washington, Seattle

Zhongsheng Zhang

Department of Biochemistry, University of Washington, Seattle

Erkang Fan

Department of Biochemistry, University of Washington, Seattle

Wesley C. van Voorhis

Department of Medicine, University of Washington, Seattle

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)

Key Words: thermal shift assays • protein unfolding • protein stabilization • superoxide dismutase

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 700-707 (2009)
DOI: 10.1177/1087057109335749


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