13
Thermal Shift Assay
Ligand binding changes the temperature at which a protein
unfolds when heated — this is the principle behind thermal shi
assay (TSA). It's a rapid and inexpensive means of screening for
binding, because it can be performed with a simple qPCR system
on up to thousands of compounds per day.
Changes in unfolding behavior are detected by monitoring
the binding of fluorophores to hydrophobic portions of a
protein which become exposed as the protein denatures.
A shi in melting temperature indicates a binding event
or hit. Various factors affect the magnitude of the thermal
shi besides the affinity of the ligand at the melting
temperature. The thermodynamics of protein unfolding
play a major role, and varies for different proteins
20
.
Although TSA requires only small amounts of protein,
the weak binding typical of fragments may not rise to
the level of detection by this technique. Because both
false positives and negatives are common occurrences,
use of TSA with larger compounds is more practical
9
. For
crystallization, this technique has utility in identifying
ligands that stabilize a protein
15
and for screening suitable
stabilizing buffers
24
. Most importantly, TSA doesn't truly
provide quantitative data on binding affinity of ligands,
meaning that calculation of K
d
is not possible using these
indirect methods.
Straightforward, label-free and in
solution measurement of changes in
melting temperatures due to binding
events
Only provides a qualitative assessment
of interactions (K
d
isn't calculated), not
ideal for weak interactions common of
fragments
