Biomedical Engineering Reference
In-Depth Information
NMR can still provide useful information through interaction and ligand-
binding studies, enabling work with larger molecular weight proteins thanis
possible for full structure determination. Particularly useful is the ability of
NMR to provide data on ligand-binding sites, kinetics and binding equilibria,
making it a valuable technique in drug research. Guided by the large
proportion of drugs already targeting GPCRs, NMR ligand-binding studies
applied to mammalian membrane proteins could likely form an instrumental
part of future drug development in this area.
A range of different NMR techniques are available for such studies
(reviewed in Yanamala et al.
286
). Recent studies have used saturation transfer
difference (STD) experiments in which a saturating proton pulse is appliedto
the receptor of interest. Magnetisation is transferred onto the ligand in the
bound state and exchanged into the free ligand present in excess, leading to
increased experimental sensitivity. Only small amounts of protein (pM to mM)
are needed, enabling NMR studies of proteins expressed at low levels, typical
for many GPCRs. STD was used to determine binding between sweet proteins
and membrane preparations of the brazzein receptor, a family 3 GPCR.
287
The
STD approach was also used in a recent study investigating the interaction of
leukotriene B4 with E. coli-expressed, amphipol-solubilised BLT2.
288
2D
NOESY spectra demonstrated that in the bound state, LTB4 folds into a
'seahorse' conformation, which differs markedly from the solution conforma-
tion, indicating it is significantly constrained by the protein environment.
Concentrations of the BLT2 receptor were low ca. 15-20 mM, whilst LTB4
ligand concentrations were ca. 140-150 mM, emphasising the benefits of
techniques which observe small ligands rather than the high molecular weight
membrane proteins to gather pharmacological and structural information.
288
Further information has been gained in a number of ligand binding studies
via the transferred NOE technique. For example binding of a PGH
2
mimic,
U46619, to unlabelled thromboxane A
2
receptor (TP) expressed in baculovirus
was investigated.
289
The free ligand tumbles rapidly resulting in negative peaks
in the NOESY spectrum, however, on addition of the protein, tumbling is
reduced and positive peaks are observed. Intramolecular NOEs between the
U46619 protons detected a conformational change on TP binding indicating
that U46619 binds in a rectangular conformation, enabling modelling of this
compound in the TP binding pocket.
289
The trNOE method was also used in
the study of the binding of S-metacholine and (2S,4R,5S)-muscarine to the M2
muscarinic acetylcholine receptor, expressed in Sf9 insect cells, enabling
determination of the binding conformation and modelling of the complex.
290
In both cases, advantage was taken of the ability to express functional proteins
via the baculovirus system, without the need for costly labelling of the target
protein. A similar approach was used in studying binding to the PACAP
receptor, a member of the larger glucagon/secretin (type B) GPCR family.
291
In a further study, trNOE data was combined with RDC measurements in
order to determine the conformational change on binding of an analogue
of the C-terminal undecapeptide from the G-protein transducin to the MII
