Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands.
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.
Crowther
Gregory J
GJ
Department of Medicine, University of Washington, Seattle, WA 98195-7185, USA. crowther@u.washington.edu
Napuli
Alberto J
AJ
Thomas
Andrew P
AP
Chung
Diana J
DJ
Kovzun
Kuzma V
KV
Leibly
David J
DJ
Castaneda
Lisa J
LJ
Bhandari
Janhavi
J
Damman
Christopher J
CJ
Hui
Raymond
R
Hol
Wim G J
WG
Buckner
Frederick S
FS
Verlinde
Christophe L M J
CL
Zhang
Zhongsheng
Z
Fan
Erkang
E
van Voorhis
Wesley C
WC
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002021:Buffers; D008024:Ligands; D010961:Plasmodium / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry*; D054852:Small Molecule Libraries / Q000032:analysis*; D044366:Transition Temperature
D002021:Buffers; D008024:Ligands; D015800:Protozoan Proteins; D054852:Small Molecule Libraries