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solvent 1 H 2 O. However, for the latter sample, expression is done in a 1 H 2 O-based
medium that is deficient in glucose and instead contains deuterated amino acids from
an algal lysate. Subsequent exchange of the purified sample into D 2 Oa lows
simplification of the amide spectrum, with only protected site remaining visible.
Since the NMR spectral properties of micelle-embedded core regions of membrane
protein structure tend to differ from solvent-exposed regions, use of these two labeled
samples allows assignment strategies to be specifically tailored to each region.
4.1.2 Methyl Protonation
Despite the utility of perdeuteration in the assignment of backbone chemical shifts,
the elimination of all but the exchangeable protons impedes structural studies that
rely on conventional NOE-based approaches. Although in some cases it is possible
to use only amide proton NOEs to obtain a protein global fold, the accuracy of these
structures tends to be low due to the small proportion of distance restraints between
protons from non-sequential residues (e.g., 5-8 ˚ backbone pairwise rmsd to target
structure [ 251 , 252 ]). Therefore to increase the number of protons in the protein
core while maintaining the benefits of extensive deuteration, a number of methods
have been developed to retain protons at specific non-exchangeable sites using a
“reverse isotope” labeling approach [ 20 , 248 , 249 ]. Methyl groups have been the
principal targets for selective protonation since they are enriched in protein hydro-
phobic cores [ 253 ], making them structurally informative sources of NOE-based
restraints [ 251 , 254 , 255 ]. In addition, rapid rotation about the methyl symmetry
axis causes its three protons to give rise to a single peak with narrow 1 H line widths
that are additionally narrowed for methyls that terminate flexible amino acid side
chains [ 256 ]. In the context of a slowly tumbling macromolecule, this rapid methyl
rotation also creates ideal conditions for the optimization of relaxation in HMQC-
type experiments (methyl-TROSY, described in Sect. 4.2 ).
A widely used strategy for the selective introduction of methyl protons into
deuterated proteins is known as the ILV method, since targeted methyl groups
reside in the amino acids Ile, Leu, and Val (reviewed in [ 256 - 258 ]). In this method,
deuterated
-keto acid precursors retaining protons at methyl sites are added to a
bacterial expression culture growing in D 2 O minimal media approximately 1 h
prior to induction of protein expression (Fig. 5 )[ 259 , 260 ]. To maintain a high
background of deuterium incorporation, uniformly deuterated glucose is included
in the growth as the only other source of carbon. After a relatively short induction
period (~4-6 h) designed to maximize incorporation and minimize metabolic
scrambling [ 259 ], methyl protons from
a
a
-ketobutyrate and
a
-ketoisovalerate will
be incorporated into the Ile(
1), and Leu/Val methyl groups, respectively. There is
a large variety of isotope label combinations available for these
d
-keto acids [ 258 ];
those shown in Fig. 5 have been the most commonly used for the study of large
proteins [ 257 ].
The ILV approach has been applied to structure determination of membrane
proteins from the
a
b
-barrel family, as first shown for OmpX in DHPC [ 261 ].
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