9
There are a few advantages of using
ligand-observed techniques. One is
that isotopic labeling of the protein
is not required. Secondly, the
spectra obtained can confirm that
both the protein and the fragment
are intact.
There are also a handful of
disadvantages. First, tens of
milligrams of protein are typically
required. Additionally, there is the
danger of weak non-specific binding
(NSB) giving rise to false positives
because of the high concentration
of ligands used. Lastly, none of the
ligand-observed techniques provide
information on the binding site.
Temperature-Related Intensity
Change (TRIC)
TRIC is particularly suited for fragment-based
screening because it can measure low affinities
and doesn't require large amounts of target or
fragments. This makes screening campaigns
more affordable for pharma and attainable for
academic or government-funded institutions.
TRIC — one of the two components of MicroScale
Thermophoresis (MST) — measures and quantifies
changes in fluorescence intensity during a laser-
induced temperature change
1.12.19
. The fluorescence
detected comes from labeling the target protein with
a suitable fluorophore or its own intrinsic tryptophan
and tyrosine residues.
When a binding event occurs between a target
protein and it's ligand, the fluorophore's chemical
