Biomedical Engineering Reference
In-Depth Information
Despite such technical limitations, RDCs have proved important in
structure determination of a number of membrane proteins including
Vpu,
225
Pf1 coat protein,
226
MerF,
227
pentameric phospholamban,
18
OmpA,
219
KcsA,
228,229
DAGK,
17
and DsbB.
16
Recently, DNA nanotubes
have been used to measure RDCs for the mitochondrial transporter, UCP2.
230
In combination with fragment searches of the PDB
231
and a limited number of
PRE restraints, this was used to define the backbone structure, demonstrating
the potential of this strategy.
12.5.3 Chemical Shift Prediction
Recent studies have demonstrated that chemical shifts present a very powerful
source of structural information that may be used as restraints in structure
calculations. Prediction of protein structures from knowledge of backbone
chemical shifts alone, combined with appropriate molecular mechanics force
fields has been demonstrated.
232,233
These methods rely on the creation of
databases of chemical shift values for known structures which can then be used
to search for protein fragments with similar chemical shifts to those of an
unknown target, enabling assembly of a backbone structure. Such methods
have proved successful for globular proteins, including for analysis of protein
complexes.
234,235
However, a limiting factor is the extensive chemical shift
information required for accurate structure prediction, which may be
particularly hard to gather for large membrane proteins where full assignment
may in some cases remain beyond reach. Consequently, it has been proposed
to combine chemical shifts with other structural restraints such as RDCs and
NOEs to both improve the accuracy of the chemical-shift-based predictions
and enable the use of reduced datasets.
236-239
Although each restraint class on
its own is unlikely to be sufficient for full structure determination, the
combination should allow high accuracy and applications to membrane
proteins are eagerly anticipated. To date, most work has involved backbone
chemical shifts. Side-chain shifts are inherently more difficult to analyse due to
the small variance in the
1
Hand
13
C methyl shift values; dynamic effects of the
side-chains, especially for solution-exposed residues, which for many of the
smaller proteins studied make up a large proportion of the available data;
relatively limited availability of side-chain assignments compared to backbone
assignments; and limited understanding of the structure and dynamics of side-
chains.
240
Nevertheless, recent developments in understanding the processes
affecting side-chain chemical shifts suggest that inclusion of this information is
possible, demonstrated by the development of the CH3Shift method
240
for
prediction of protein methyl chemical shifts and it is expected that with
increases in the available side-chain chemical shift data in the BMRB, as well
as developments in the molecular mechanics force-fields for side-chains, the
accuracy of such approaches will improve.
