Chemistry Reference
In-Depth Information
these methods will in general increase with increasing size of the target protein. Two main
classes of ligand detected NMR binding assays can be distinguished:
1. methods that rely on transfer of magnetization between target and ligand;
2. methods that rely on the detection of an altered hydrodynamic property (i.e. molecular
tumbling rate or diffusion rate) upon binding to the target.
Methods that represent the first class of experiments are saturation transfer difference
(STD) [ 72 ] and WaterLOGSY. [ 73, 74 ] These techniques are closely related in that they rely
on dipole-dipole interaction between ligand and target protein spins. Diffusion and relax-
ation filter-type experiments belong to the second class of methods. [ 75, 77 ] When a small
ligand binds to a macromolecule, its apparent rates of translational diffusion and reorient-
ation are slowed. The transferred NOE technique [ 78, 83 ] also falls into this class. It relies
on detecting intra-ligand NOEs that develop in the bound state, where the dipole-dipole
interaction due to the decreased molecular tumbling rate is much more efficient than
in the free state, by observing the free ligand. Experiments that do not fall in either
category are the use of paramagnetic spin labels to increase the relaxation of nearby
spins [ 81, 82, 84 ] and fluorine NMR to detect binding or displacement of fluorinated com-
pounds to a target. [ 85 88 ] Selected ligand-detected NMR techniques are presented in more
detail below.
In the ligand-detected NMR techniques, the ligands are usually present in molar excess
compared with the target protein in the samples. As always when detection of binding
depends on exchange between the bound and free states of the ligand, a minimum off-
rate ( k off ) will be required for detection of binding. This should not pose a problem for
fragments, however, since they are expected to have diffusion controlled on-rates ( k on ) and
will not bind with very high affinities. Alternatively, a 'spy' molecule binding specifically
at the binding site of interest can be employed. [ 45, 76, 86, 89, 90 ] The binding signals of the
spy molecule are observed and ligands that are able to displace the spy molecule will be
detected as binders. There are distinct advantages with using a spy molecule and indirect
detection of binding via competition experiments for screening: there is no minimum off-
rate necessary for detection of binding and no purely nonspecific binders will be picked up.
On the other hand, the requirements for the spy molecule are high: it has to bind specifically
to the desired site with a low enough affinity to be readily displaced by weakly binding
fragments. The detection cut-off for the fragment affinity will be determined by the affinity
of the spy molecule to the target protein. Further, it must display a clear binding signal
in the chosen NMR technique and preferably cover the entire binding pocket of interest.
A drawback is that no fragments binding to adjacent subsites to the binding site of the spy
molecule will be detected. Such fragments could be of interest for linking or expansion of
the fragment hit.
Saturation transfer difference. The origins of the STD experiment [ 72 ] can be traced to
the spin-saturation transfer experiment or Forsén-Hoffman experiment from the 1960s. [ 91 ]
In the STD experiment, a subset of the protein 1 H resonances are saturated by means of
a train of frequency-selective radiofrequency pulses applied to a narrow spectral region
devoid of ligand resonances. The saturation is transferred by spin diffusion ( 1 H- 1 H cross-
relaxation pathways) to the rest of the protein, a process that becomes more efficient with
Search WWH ::




Custom Search