Chemistry Reference
In-Depth Information
the membrane proteins. An appropriate choice for such a reference protein would be one
with few known binders, in order to minimize the chances of nonspecific binding. The
E. coli
Outer membrane protein A (OmpA) was chosen for such qualities. This reference
protein was of similar size to our intended target and also refolded in DPC micelles. To get
an initial feel for whether we could detect specific binding to a membrane protein using
TINS, we conducted a proof of principle study with a screen of a small subset (about 100
compounds) of our compound library using KcsA from
Streptomyces
as the target and
OmpA as the reference.
Prior to screening, it was necessary to establish an appropriate (1) level of DPC to include
in the wash buffer to maintain the integrity of the immobilized, micelle solubilized target
and (2) internal reference compound. If the DPC concentration in the environment of the
target decreased to below its CMC, the micelles formed by DPC would start to dissoci-
ate slowly into monomers and be flushed away. Simple calculation suggested that it was
necessary to use DPC at 5mM in the wash buffer to in order to maintain the concentration
above the CMC (1mM) upon dilution with the compound mix with DPC absent. We tested
both TSP and TMA as a possible internal standard by including both in a mixture with
(4-fluorophenyl)methylsulfanylmethanimidamide (FPMSMA) (Figure 6.6A), a known
(a)
H
H
(b)
1
5
HO
N
N
H
H
3
C
H
3
C
H
3
C
O
O
H
3
C
OH
H
S
HN
S
CH
3
H
O
1
H
H
H
H
2
3
4
F
5
TINS
8
6
4
2
[ppm]
Figure 6.6
Proof of principle ligand screen against a bacterial membrane protein.
(A) Structures of the known ligand (4-fluorophenyl)methylsulfanylmethanimidamide used to
determine the integrity of the immobilized KcsA. (B) Detection of ligand binding in one mix
during the screen. A mix containing five different compounds was applied simultaneously
to the cell containing immobilized KcsA and to the cell containing OmpA. The individual
1
H
NMR spectra of each cell are overlaid (labeled TINS). The
1
H
NMR spectrum of each
individual compound, which has been intentionally line broadened to approximately match
the linewidth of the TINS spectra, is shown above (numbered). All peaks from compounds
1
and
5
were reduced in amplitude in the presence of the immobilized KcsA with respect to
OmpA, indicating that these compounds bind to KcsA. The structures of compounds
1
and
5
are shown.
