Practical Aspects of Using NMR in Fragment-based Screening - Fragment-Based Drug Discovery - page 71

Fortuitous ligands. Ligands from the expression host (e.g. fatty acids from Escherichia
coli ) could be noncovalently bound, e.g. in the active site of the overexpressed target
protein. This is a serious, but rare, problem and may require an additional refolding step in
the protein purification protocol. The presence of any noncovalently bound ligands in the
target protein can be checked by nondenaturing ESI-MS.

4.5 NMR Techniques for Fragment-based Screening

NMR spectroscopy is a versatile tool for assaying binding of a small molecule to a mac-
romolecule such as a target protein. There are many excellent reviews describing NMR
techniques used in fragment screening. [ 6, 70, 71 ] The available techniques may be divided into
two different classes, ligand-detected and protein-detected methods. The protein-detected
methods yield more information on the ligand-protein interaction, but the ligand-detected
methods are more widely used in the pharmaceutical and biotech industries since they can
be applied to amuchwider range of target proteins and require less upfront resources. There-
fore, this part will focus on the ligand-detected methods. Examples of NMR techniques for
monitoring protein-ligand interactions are given in Table 4.1.

Table 4.1 Examples of NMR techniques for monitoring protein-ligand interactions.

NMR technique
or observable

Nonbinding
ligand

Protein Bound ligand Representative

references

Saturation
transfer difference

Unsaturated Saturated Saturated 72

WaterLOGSY Positive NOE Negative NOE Negative NOE 73, 74

Linewidth Narrow Broad Broadened 43

Transverse
relaxation
filter/selective
longitudinal
relaxation

Slow Rapid Increased 75, 76

Translational
diffusion

Rapid Slow Slowed 75, 77

Transferred NOE Weak

positive NOE

Strong
negative NOE

Weak negative
NOE

78-80

Paramagnetic
spin label

Slow
relaxation

— Rapid relaxation 81, 82

Protein chemical
shift perturbation

— Unshifted Shifted 32

4.5.1 Ligand-detected Techniques

The ligand-detected techniques rely on detecting changes in the NMR observables of a
ligand upon exposure to the target protein. The ligand will transiently adopt NMR para-
meters characteristic of the typically much larger target protein. Hence the sensitivity of