Chemistry Reference
In-Depth Information
immobilized and, in comparison with nonimmobilized receptors, the immobilized recept-
ors appear significantly more stable. At present the density of receptors is insufficient to
perform NMR ligand screening, but work is in progress to address this issue.
6.3.4
Screening
We have developed a diversity library for use in TINS and it is our intention to screen it
against all targets. The design requirement for high solubility (to maximize oral bioavailab-
ility) pays dividends when used in membrane protein ligand screening, since partitioning to
the lipid phase is minimized. Nonetheless, as with soluble proteins, it remains important to
use an appropriate reference system to cancel out nonspecific binding events. We have used
the
E. coli
protein OmpA as a successful reference protein in one partial screen of about
200 compounds and one complete screen of about 1300 compounds. Its advantages include
easy expression and purification, solubility in DPC and low small-molecule binding. One
potential way to avoid the use of a reference protein would be to screen using a known,
competitive ligand. We are currently adapting the hardware of the TINS ligand screening
station to permit competition ligand screening studies. In this arrangement, the target is
immobilized in both cells of the sample holder and the same mix is applied to both cells
whereas the competitor is added to only one of the cells. Competition ligand screening
will eliminate the need for a separate reference protein but has the drawback that one can
only find ligands to known binding pockets. When it becomes possible to screen proteins
in native membrane vesicles, then a preparation of membrane vesicles of parental cell lines
not expressing the target should serve as an ideal reference.
In order to improve the robustness of TINS further, we include a reference compound
in all mixtures that can be used to scale the two spectra post-acquisition. With membrane
proteins, even more so than with soluble proteins, it is important to ascertain whether the
reference compound interacts with the target or the surfactant used to solubilize it. The ideal
reference compound has only one peak outside the spectral range of all compounds and,
naturally, does not interact with the reference, target or surfactant. TSP fulfils most of these
requirements but does bind to some targets. Alternatives that we have used include glycine
and tetramethylammonium chloride (TMA). A crude scaling factor for the two cells can be
determined experimentally by integrating the water signal from each cell using a standard
1D imaging experiment with a single scan. Binding of potential reference compounds can
readily be established by simply conducting TINS experiments on all, applying the scaling
factor and analyzing the spectra for equal peak intensity in both cells. So far we have not
encountered a case where more than one of the three potential reference compounds bound
to the target.
As noted previously, individual detergent molecules rapidly exchange between the
micellar and monomeric forms. Thus, washing of immobilized micelles in buffer without
detergent leads to rapid loss of protein functionality, as shown in Figure 6.5. At least for the
case of KcsA, which consists of a single polypeptide, the loss of functionality (as measured
by binding of a known ligand) appears to be perfectly reversible. Nonetheless, it is clear
that DPC must be applied throughout the screening procedure. Since DPC is available in
deuterated form, its presence does not interfere with the acquisition of the NMR spectra of
the compounds. For convenience we chose to include DPC only in the buffer used to wash
the compounds out of the cells of the sample holder and not in the mixes themselves. Since
