Chemistry Reference
In-Depth Information
NMR parameter that occurs upon their interaction [15]. The more common NMR
screening techniques used to identify protein-ligand binding affinity are focused
on the observation of either protein NMR parameters or ligand NMR parameters.
The choice of which path could be used depends on certain conditions, such as the
macromolecular target size, availability of protein by recombinant techniques and
the K
D
complex [1].
At first, all NMR spectroscopy parameters might be able to determine protein-
ligand binding; however in practice only parameters easily obtained and highly
sensitivity are applied [3]. When protein-based NMR methods are used, the
parameter that can be observed is chemical shift changes, after and before
interaction with ligand. The greatest advantage of this methodology is that it does
not rely on fast exchange to get bound state information, allowing the
characterization of both higher and lower binding affinities. Furthermore, by
identifying perturbations of assigned chemical shift of protein it is possible to
localize and characterize the binding sites [19].
However, the major limitation of this method is the need of milligram quantities
of
15
N- or
13
C-labeled protein soluble and also heteronuclear two-dimensional
NMR correlations well-resolved [1]. Thus, in some cases, the time required for
NMR assignment of such targets is so large that other techniques, as X-ray
crystallography, are favored because they are capable of promoting high-
resolution structural information to medicinal chemistry on a faster time scale
[19]. Details of protein-based NMR methods will be further discussed.
Another possibility to determine protein-ligand binding affinity is a ligand-based
NMR method, which compares parameters of small molecules in the presence and
absence of the receptor. In this case, the NMR parameters used are more
diversified, including changes in transverse and longitudinal relaxation rates,
changes of diffusion constant, changes of NOEs, or exchange of saturation. In
turn, for this methodology it is unnecessary to utilize isotopically enriched protein
that allows to evaluate different targets more rapidly. Additionally, analysis of
signal from ligand NMR spectra is more simplified compared to protein NMR
spectra.
