Biomedical Engineering Reference
In-Depth Information
usefulness is demonstrated by the recent rise in crystal structures of G-protein-
coupled receptors (GPCRs),
4,20
their application to NMR studies, including
enabling advanced isotope labelling, is costly and in many cases frequently
requires further development. Nevertheless, promising progress in this regard
suggests that a wider range of expression systems will be available for NMR
studies in the future.
11,21,22
Obtaining sufficient structural restraints for 3D
structure determination is a limiting factor, particularly when relying on
traditional NOE distance restraints. Advanced isotope-labelling schemes are
presented which have proved highly successful in a number of cases. In
addition a range of other technologies are available to obtain such distance
restraints; some such as paramagnetic relaxation enhancement (PRE) and
residual dipolar coupling (RDC) restraints have already been demonstrated on
membrane protein targets, whilst others, such as pseudo-contact shift (PCS)
restraints show considerable promise but as yet are limited to studies on
soluble proteins.
Most NMR studies of membrane proteins have been carried out in detergent
micelles. Despite the relatively large size of protein-detergent micelle
complexes, they are much smaller than more native-like membrane mimetics
and thus give more favourable NMR spectra. Questions about the relevance of
the micellar environment to mimic the membrane surroundings of a given
protein remain in many cases. A number of other media have been developed
23
including those which are believed to be a much closer mimic of a cellular
membrane entity.
24
The potential for NMR to study membrane proteins in a
more native environment has been demonstrated by a number of recent studies
in such media.
24-26
As always, the functional integrity of a protein in its
membrane mimetic needs to be tested by a suitable assay.
With hundreds of members, easily forming the largest protein family in
eukaryotes, GPCRs are estimated to comprise around 30% of current drug
targets.
27
Until recently, very little structural information about these seven
transmembrane-helical receptors was available. However, the recent publication
of crystal structures of several class A receptors, some of them captured ina
range of conformational states linked to different levels of activity,
4
indicate that
X-ray crystallography has matured to a level where such structure determination
is now becoming more routine. These impressive structures of GPCRs are the
result of a multitude of incremental improvements to the experimental protocols
and techniques used. Crucially, in all cases, issues related to inherently low
protein stability and mobility of loop regions were tamed by various protein
engineering approaches facilitating better crystallisation.
Recent developments, in particular the solution NMR structure of an
archaeal seven-transmembrane receptor
19
indicate that under favourable
conditions, structure determination of similarly stabilised members of class
A family GPCRs, comparable in size to sensory rhodopsin, might also be
feasible. Nevertheless, whilst NMR structures of GPCRs are eagerly
anticipated, the range of functional data that could be provided by NMR
studies is likely to make a much greater contribution to this field. As discussed
