Chemistry Reference
In-Depth Information
Consequently, a disadvantage of ligand-based NMR methods is the incapacity to
localize the binding site into the receptor. Moreover, it relies on fast exchanges
system which transfers bound state information to the free state,
i.e.
weakly
binding ligands are observed, whereas ligands with high affinity will be missed.
This requirement leads to the use of large ligand molar excesses, and the
consequent risk is that, under these conditions, ligand may start to occupy weaker
affinity nonspecific binding sites. Recent advantages and more details in ligand-
based methods are discussed below.
Protein-Based NMR Methods
Protein-based NMR methods are based on the chemical shift difference in the
resonance signal of the protein atoms when they are interacting or not with the
ligand. This methodology relies on the fact that protein chemical shifts are
sensitively dependent on both chemical environment of the respective residue and
conformational/dynamic changes upon ligand binding [18].
Considering the
difficulty to analyse
1D protein spectra due to signal overlapping,
HSQC
(heteronuclear single quantum coherence) and TROSY (transverse relaxation
optimized spectroscopy) [20] experiments are frequently the most used. However,
15
N-labeled protein is required
to perform
these techniques.
All amino acids in
15
N-labeled protein, except proline, give rise to single peaks in
a
1
H-
15
N HSQC or TROSY spectrum [18]. For ligand binding studies, the NMR
spectrum is usually obtained in the absence and presence of a ligand, which is not
observed
since it is not
15
N-labeled. Protein chemical shifts changes indicate
binding. Thus, it
can be used to
identify the
location of binding sites
in the protein
[14].
1
H-
15
N HSQC experiments are limited to smaller proteins (<35 kDa), but the
introduction of transverse relaxation optimized spectroscopy (TROSY)-type
experiments potentially enables the study of larger protein targets [7].
In some cases, an alternative to
15
N,
1
H-HSQCs using
15
N-labeled protein has
been suggested to record
13
C-
1
H correlation spectra with
13
C-methyl-labeled
protein [21]. On the other hand, to overcome the shortcomings of
1
H/
13
C-HSQC
for screening, another technique was suggested to allow only the selective
