Chemistry Reference
In-Depth Information
In all cases, the flipped poses had significantly higher COST, typically greater than 50%
more than the lowest COST poses. We are in the process of examining whether local
interactions observed between Bcl-x
L
and
7
are reproduced by the low COST structures.
5.8 Applications to Fragment-like Compounds Bound to Large
Proteins
5.8.1 Nonuniform Protein Labeling
In its initial embodiment,
[
15
]
NOE matching was designed for unlabeled compounds bound
to uniformly protonated,
13
C/
15
N-labeled protein samples. The initial success of NOE
matching is due to the fact that, even in the absence of protein assignments, the
pat-
tern
of NOEs contains enough information to limit the number of residue types that need
to be considered as the potential partner giving rise to the NOE. As the size of a pro-
tein becomes larger, sensitivity is dramatically reduced due to increased relaxation rates,
yieldingmany fewer NOEs and limiting the information content that can be extracted. How-
ever, protein-ligand NOE interactions with high sensitivity and information content can be
obtained by nonuniform isotopic labeling schemes. For example, specific types of amino
acid residues that are isotope labeled in a particular manner (e.g. protonated or proton-
ated and
13
C/
15
N labeled) can be incorporated into an otherwise uniformly perdeuterated
(or perdeuterated and
[
15
]
N labeled) protein background.
[
35 38
]
These procedures produce
protein samples that are labeled by residue type (residue type specific labeling). Residue
type and residue type/atom type specific labeling schemes yield enhanced NOE sensit-
ivities and, by reducing spin diffusion, more accurate distance restraints. These labeling
schemes have been used to observe protein-ligand NOEs for complexes involving large
proteins.
[
39, 40
]
With regard to NOE matching, selective labeling schemes are important in that they can
provide the identities of the residue types involved in protein-ligand NOEs. Furthermore,
when a particular residue type occurs only once in a binding pocket, residue-type specific
labeling combined with protein-ligand NOE experiments directly provide sequence-
specific resonance assignments. An approach using a series of residue-type specific labeled
samples in conjunction with saturation transfer difference (STD) NMR experiments has
been described for characterizing ligand binding poses (SOS NMR
[
41
]
). Compared with
an STD spectrum, a 2D or 3D NOESY spectrum using a residue type specifically labeled
sample contains significantly more information. For example, given a valine-type specific
labeled sample, observation of an STD only indicates that
one or more
protons on
one or
more
valines is/are close to a given ligand proton. The NOESYdata provide information on
how many valines
and
which specific valine atoms
(methyl, HB, HA) are close to a given
ligand proton. NOE matching utilizes the additional information obtained in a NOESY
spectrum.
In an effort to extend the applicability of NOE matching to the larger proteins of pharma-
ceutical interest, for example kinases and phosphatases, we have adapted NOE matching to
be able to utilize data from any residue (and atom)-specific labeling scheme. An illustrative
example is provided below.
