Biomedical Engineering Reference
In-Depth Information
complexes
and
require
low
protein
concentrations.
A
wide
variety
of
techniques has been developed over the years.
Saturation transfer difference (STD) developed by Mayer and Meyer
20
was
one of the first methods proposed for ligand-observed screening of fragment
libraries and remains one of the most popular. This method consists of
selectively saturating a resonance of the protein. The saturation is efficiently
spread over the entire protein via spin diffusion and if a ligand binds, the
saturation is propagated from the protein to the compound by cross relaxation
at the ligand-protein interface. This method requires a large excess of ligand
and allows measurements using a low protein concentration. STD can also be
used to obtain structural information on the complex using a technique known
as epitope mapping
21
(see below). Although widely used, STD does suffer from
being sensitive to the size of the target (where bigger proteins are better),
artefactual magnetisation transfer and a limited affinity range (10 nM , K
D
,
1-2 mM).
22,23
Typically, STD is used in combination with other ligand-
observed techniques such as WaterLOGSY (see below) to eliminate false
positives from screening data.
24
One interesting modification to STD, called Group Selective-STD, directly
saturates certain classes of
1
Hs of the target (e.g., amide) in a selective manner.
The method avoids the dependence on spin diffusion which is inefficient for
proteins of molecular weight less than 20 kDa. For
15
N-labelled targets, an
elegant manner of directly saturating the amide
1
Hs has been developed.
25
In
order to ensure that saturation is absolutely specific for the target, the method
uses a half-filter to eliminate magnetisation from
1
Hs not attached to
15
N
spins. However, the required KJ
15N1H
delay may reduce sensitivity due to
relaxation. When the ligand resonances do not overlap with the amide region
of the protein, the half-filter can be removed to improve sensitivity. Although
perhaps not ideal for screening large libraries of ligands that will inevitably
contain resonances near those of the amide protons of proteins, the method
has been put to good use to elucidate protein-ligand structures (see below).
26
One of the reasons for the popularity of STD is the ability to apply it to a
wide variety of systems to detect ligand binding. Recent applications of STD
include lipopolysaccharides,
27
glycoproteins
28
and nucleotide sugar transpor-
ters.
29
Perhaps the most interesting and challenging application has been to
detect binding of ligands to membrane proteins, either purified GPCRs
30
or in
live cells.
31
Water-ligand Observed via Gradient SpectroscopY (WaterLOGSY) is a
commonly used alternative to STD.
32
Based on the observation that ligand-
binding sites frequently also contain bound waters, the technique makes use of
the abundance of water to efficiently transfer magnetisation to a ligand in close
proximity via multiple pathways. As the method is very sensitive, requires low
protein and modest ligand concentrations, it has found frequent application in
fragment-based drug discovery. However, one has to take care that the
magnetisation is not transferred to the ligand via an artefactual mechanism
such as chemical exchange and therefore, as with STD, other ligand-observed
