Chemistry Reference
In-Depth Information
immobilize His-tagged protein using Zn
2
+
instead and leaching does not pose a problem.
However, despite the fact that a Sepharose resin is used in conjunction with a diamagnetic
ion, there appears to be additional line broadening effects (Figure 6.2). These may result
from nonspecific interactions with available NTA sites on the resin which turn out to be
difficult to block.We have also used streptavidin Sepharose to immobilize biotinylated ribo-
nucleotides for ligand binding studies. This system is convenient and yields high quality
NMR spectra. By blocking unoccupied binding sites with free biotin (and naturally using
streptavidin Sepharose as the reference sample) one should be able to limit small-molecule
binding to sites that are not on the target; however, we have not carried out a full screen on
such a system so it not possible to make a definitive statement at this time. Other affinity
tags can also form the basis of successful, NMR-compatible immobilization. For example,
Haselhorst
et al
. have recently reported the use of Strep-tactin Sepharose, a variant of
streptavidin Sepharose, to perform saturation transfer difference (STD) studies.
[
19
]
6.2.3 Ligand Screening
We decided to carry out our ligand screening studies using mixes of compounds at a
very early stage in the process of developing TINS. This decision was made on the basis
of throughput and robustness. Since our mixes consist of on average five compounds,
obviously throughput is increased by a factor of five with respect to screening singletons.
Also, since it is expected that only one compound (and occasionally two) per mix bind to
the target, most peaks in the reference and target spectra should be of the same amplitude.
If this is not the case, it may be a sign that there is a problemwith the screening sample. The
use of mixes requires a strategy to design them properly. Given the constraint of increased
linewidth generated by the heterogeneous TINS system, the primary factor governing the
selection of compounds for a mix is the number of well-resolved peaks for each. We have
therefore recorded a reference 1D
1
H spectrum of every compound in the ZoBio/Pyxis
fragment collection at 500 μM in phosphate-buffered saline (PBS) in the presence of a
fixed amount of TSP. The reference spectra also serve the dual role of quality control.
The reference spectra are automatically peak picked and the peak positions stored in our
database.We have developed an in-house algorithm to select compounds randomly from the
collection and test them rapidly for TINS compatibility, that is, at least three well-resolved
peaks for each compound (when available). This allows us to read out the ligand from the
mix directly without further deconvolution (see below). The algorithm also places explicit
limits on the number of aromatic compounds per mix and avoids mixing compounds with
p
K
a
extrema. Once designed, the mixes are then made at 500 μM for each compound in
PBS. The mixes are stored at room temperature and subsequently inspected visually for
signs of precipitation. About one-third of mixes are rejected at this point. Mixes that do
not precipitate are subjected to
1
H NMR analysis, where we expect to see that the NMR
spectrum of the mix is a simple sum of the NMR spectra of the individual compounds using
TSP as a reference. Changes to the NMR spectrum of the mix, which we rarely observe,
are indicative of possible aggregation behavior of the compounds.
In order to carry out a ligand screen, the resin bearing the target and reference proteins,
which have been immobilized at a solution equivalent of about 100 μM, must be packed
into the dual-cell sample holder. A home-made packing reservoir has been built to fit on
top of the dual-cell sample holder and double the volume of each cell. The resin (as a 50%
