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of spectra that can be attained. When high quality spectra of relatively small
detergent-protein complexes are available, conventional NOE-based approaches
can be applied in a straightforward manner without the need for deuterium labeling
or TROSY type experiments. This tends to be the case for structures of single TM
helices in monomeric or dimeric forms, examples of which are shown in Table
2
,
Method I, Fig.
6a
. Structures of larger oligomeric states formed by single TM
helices have also been determined in this way, although some
2
H-labeling and
TROSY-based backbone assignment experiments were required to deal with the
larger size of these complexes (Table
2
, Method II, Fig.
6b
).
When the length of the polypeptide exceeds that of these single TM-helix
constructs, TROSY-based experiments and uniform deuteration are usually used to
obtain backbone assignments. In cases where spectral quality or complexity prevents
straightforward assignment of backbone resonances from uniformly
15
N,
13
C,
2
H-
labeled samples, it is possible to increase the number of assignments with samples
selectively
15
N- or
15
N,
13
C-labeled with a single amino acid. For example, ~20
different selectively labeled samples were used to help assign backbone atoms in the
human mitochondrial VDAC
-barrel [
73
]. Alternatively, if the complex being
studied is comprised of more than one polypeptide chain, backbone assignment can
be assisted by combining labeled and unlabeled subunits, as was done for the
heterotrimeric natural killer cell-activating complex [
65
]. Additional assignments
are also sometimes accessible through the use of a range of temperatures [
184
]orpH
conditions [
40
] that allow observation of different subsets of resonances. As a last
resort it is also possible to use mutagenesis [
273
,
297
,
298
], although this approach is
not practical for the assignment of a large number of residues.
In cases where spectral quality allows a high level of backbone assignments to be
made, it is usually possible to acquire assignable NOEs between amide protons. For
b
b
-barrel folds these NOEs can be used with backbone torsion angle input derived
from secondary backbone shift data [
299
], and hydrogen bond restraints inferred
from amide solvent exchange to construct an informative global fold (Table
2
,
Method III, Fig.
6c
)[
68
,
69
,
72
]. Selectively methyl labeled ILV has also been used
for this fold type as an additional source of NOEs in some cases (Table
2
,
Method IV, Fig.
6c
)[
70
,
71
,
73
]. For helical membrane proteins, more NOEs are
required to obtain a structure of comparable precision (as described in Sect.
4.1.2
),
usually by extending chemical shift and NOE assignments to other side chain
atoms. This requires that 3D
1
H-
1
H correlation spectra on partially or fully
protonated samples are of sufficient quality for assignment, an achievement that
that has only been realized in a very small number of cases (see Table
2
, Method V).
5.2 Non-uniform Sampling
One of the most challenging membrane protein structures determined to a high
resolution via an NOE-driven approach is the pSRII GPCR (Fig.
6d
), which
required additional sensitivity-enhancing approaches to be employed during data
