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determination of helical integral membrane proteins has been more difficult to
realize. Even in the case of helical water-soluble proteins it was noted that
fewer long-range methyl-based restraints are available relative to other fold
types [ 251 ]. Similar studies focusing on all-helical membrane protein folds
substantiated this observation, with structure accuracies in the 5 ˚ region even
when complete assignment of methyl-associated NOEs was assumed to be possi-
ble [ 262 ]. In reality the low spectral dispersion that is characteristic of helical
membrane proteins can significantly impede chemical shift and NOE assignment
for these methyl groups, as was the case for the DAGK trimer [ 111 ]. On the other
hand, relatively high spectral quality for the pSRII GPCR allowed ~50% of
expected inter-helical NOEs involving these methyl groups to be assigned, with
the remainder being either absent from the spectrum or buried under strong
diagonal signals [ 40 ]. Yet this level of NOE assignment was still not sufficient
to generate high-resolution structures, making it was necessary to acquire a greater
number of long-range restraints involving Ala, Thr, Ile(
2), and Met methyl group
NOEs. In the case of pSRII it was possible to use the ILV methyl assignments to
help extend the chemical shift assignments to these methyl groups using a fully
protonated sample. This afforded a ~2.5-fold increase in the number of inter-
helical NOEs that could be assigned and helped to increase the quality of the
resulting structures.
When the spectral quality of a fully protonated sample is not sufficient to help
increase the number of methyl proton assignments, other methyl protonation
strategies are also available. For example, methyl protonated Ala can be directly
incorporated into deuterated proteins so long as a trio of deuterated precursors
(Fig. 5 ) that suppresses metabolic scrambling to undesired sites is also added to the
media. [ 263 ]. The direct bond between the Ala methyl group and C
g
backbone
atoms make these methyl groups excellent sources of information for backbone
structure and dynamics [ 264 ]. Importantly, the ILV and Ala-methyl labeling
strategies are complementary, allowing protons to be simultaneously introduced
to ILV and Ala methyl groups in a single sample. Ile(
a
1) and Ala methyl groups can
also be simultaneously labeled in an alternate strategy that uses deuterated rich
media supplemented with appropriately labeled Ala and a -ketobutyrate [ 265 ].
Methionine methyls can similarly be targeted by the inclusion of protonated Met
[ 266 ] or a selectively protonated
d
-keto acid derivative [ 267 ] into the D 2 O minimal
expression medium (Fig. 5 ). The C
a
g
2 proton of Ile can also be targeted by including
a
-hydroxybutyrate in the minimal media [ 268 ]. Meanwhile, selective
incorporation of protons beyond the methyl groups has been demonstrated in the
stereo-array isotope labeling (SAIL) strategy [ 269 , 270 ]. This technique uses cell
free expression to incorporate a complete suite of synthetically prepared stereo- and
regiospecifically 2 H-labeled amino acids, producing a sample with reduced 1 H
density that retains a larger number of structurally informative protons. Overall,
the general utility of these various strategies will depend on the characteristics of
each sample, as well as cost-effective availability of labeled precursors. However,
they should prove to be increasingly useful as the number of large membrane
proteins being studied by solution NMR continues to increase.
-aceto-
a
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