Chemistry Reference
In-Depth Information
this approach has led to two successful screens of membrane proteins, we are optimistic
that it will be general. In this way, it may prove possible to acquire NMR spectra even in the
presence of nondeuterated detergents, since the concentration of the monomer is reduced
by application of the compound mix in the absence of detergent. However, we have yet
to test this hypothesis. Once the immobilized protein functionality has been verified, it
is also important to create checkpoints at different time points of the screen with mixes
containing a known binder as a positive control to check that protein functionality and thus
conformation is maintained through the screen.
40
30
20
10
0
Control
1
2
3
4
5
6
No DPC
DPC
Number of mixes
Figure 6.5
Requirement for the presence of detergent while screening micelle-solubilized
membrane proteins. In this series of experiments both the target (KcsA) and the reference
(OmpA) were immobilized at a solution equivalent of 150 μM. The histogram represents the
fractional difference in peak amplitude of a known ligand of KcsA in the presence of KcsA and
OmpA. The bar labeled control represents the first application of the ligand. Subsequently three
injections of the ligand were performed using buffers that contained no detergent. A further
three injections were performed where the buffer used to wash the immobilized samples
contained deuterated DPC.
One final issue deserves special attention when considering carrying out ligand screening
studies on a membrane protein, namely the kinetics of ligand binding.Although low-affinity
ligands for soluble proteins nearly always exhibit rapid exchange kinetics on the NMR time-
scale, this may not be the case for membrane proteins. For example, histamine binds the
human H1 receptor with a
K
d
of 20 μM.
[
52
]
Such a small molecule (histamine fits well within
the definition of a 'fragment'), binding with moderate affinity would normally imply a fast
on-rate. However, in this solid-state NMR study, the on-rate was found to be of the order of
minutes! Likely mechanisms for such slow binding include access to the active site of the
protein via the membrane or slow conformational exchange of the protein due to interaction
with the membrane (or membrane mimetic). Since the dynamic behavior of detergents and
phospholipids is strongly temperature dependent, it may be necessary to carry out screening
at near physiological temperature, where the long-term stability of the target may be less
than optimal. In such situations, it may be necessary to prepare multiple samples in order
to carry out successfully a screen of a complete fragment library.
