Biomedical Engineering Reference
In-Depth Information
Finally, protein-observed NMR is commonly used to validate hits selected
via a pre-screen. A nice illustration is provided by the virtual screening of the
dishevelled PDZ domain.
11,12
The
15
N HSQC-monitored titration of 15 hits
from the screen into the labelled PDZ domain showed that all hits bound the
peptide binding site with the most potent having an affinity in the low mM
range. One of the hits blocked Wnt signaling in a cellular assay.
11.2.2 Ligand-Observed NMR
Ligand-observed NMR is the most commonly used amongst all screening
techniques as it does not require any isotopic labelling and few limitations are
imposed on the target. Ligand-based NMR techniques are mostly based on the
difference in size between the ligand and the protein. The size difference
manifests itself in numerous observables such as enhanced relaxation or a
change in the diffusion coefficient. In addition, complex formation may be
observed by transfer of magnetisation from protein to fragment by e.g.,
saturation transfer difference
20
or by changes in the chemical shift or lineshape
of
19
F spins in a ligand.
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11.2.2.1 Relaxation Methods
In NMR, relaxation is the process which restores equilibrium magnetisation.
Two different types of relaxation can be distinguished and characterised by
their rate: longitudinal relaxation and transverse relaxation. Longitudinal
relaxation is a complex function of molecular weight while transverse
relaxation increases with molecular weight. Ligand-observed NMR techniques
using relaxation methods are based upon the fact that a ligand bound to the
protein
adopts
the
relaxation
properties
of
the
complex,
i.e.,
transverse
relaxation will be greatly enhanced.
As just described, larger, more slowly tumbling molecules relax much faster
than small molecules. By extension, immobilisation of the target on a solid
support will enhance transverse relaxation by at least two orders of magnitude
relative to a small molecule in solution. We have constructed an NMR
fragment-screening apparatus based on this principle which we call TINS for
Target Immobilized NMR screening.
13
TINS uses differences in the spectrum
of the small molecule in the presence of the target and a reference protein to
detect binding. In a first, TINS was used to screen a fragment collection for
specific binding to a membrane protein (DsbB) that forms part of a disulfide
catalytic cascade in gram-negative bacteria.
14
Membrane proteins are
particularly challenging due to the very high level of false positives that arise
from non-specific binding to the hydrophobic solubilisation media. In this
case, the reference sample is used to cancel out the non-specific componentof
binding. More recently, we have also succeeded in screening a GPCR using
TINS.
15
