Chemistry Reference
In-Depth Information
protein-detergent complexes show that the detergent can add as much as 60 kDa to
the protein molecular weight (e.g., DAGK [
247
], VDAC-1 [
73
]). In order to counter
the loss of NMR signal sensitivity that is characteristic of these large complexes, it
is usually necessary to use approaches originally developed for study of large
water-soluble proteins. Those that have found the most widespread utility are
deuterium isotope sample labeling and relaxation-optimized pulse sequences.
4.1
Isotope Labeling Schemes for Large Protein Complexes
4.1.1 Deuteration
To compensate for the unfavorable relaxation properties of protons in large protein
complexes, all the non-exchangeable carbon-bound protons can be replaced with
deuterium (reviewed in [
20
,
248
,
249
]). As the gyromagnetic ratio of
2
H is 6.7-fold
lower than that for
1
H, many of the relaxation pathways that would otherwise be
present in fully protonated samples are greatly attenuated in a deuterated sample.
This leads to an increase in T
2
transverse relaxation times, and more effective
preservation of signal over the numerous coherence transfer elements that occur in a
typical multidimensional NMR pulse sequence. Significant sensitivity gains have
been demonstrated for uniformly deuterated proteins in many triple resonance
applications with both water-soluble [
20
] and membrane protein samples [
111
].
As a result, uniformly deuterated samples are now routinely used to obtain back-
bone assignments for large membrane proteins.
Since backbone assignment experiments use amide proton magnetization to
generate and detect the NMR signal, deuterated backbone amides must first
undergo complete exchange with solvent
1
H
2
O protons [
20
]. Although the extended
exposure to
1
H
2
O solutions during purification can be sufficient to re-introduce
protons at all sites, many of the larger membrane proteins that exhibit good spectral
properties are highly stable and therefore resistant to this process [
112
]. In these
cases re-introduction of protons at exchangeable sites can be extremely slow in the
core, making it impossible to achieve significant exchange without the exposure to
destabilizing conditions. This can be particularly challenging for proteins that have
been optimized to maximize stability, potentially necessitating the development of
high-yielding unfolding/refolding protocols. This was done for the 4-TM DsbB,
which had to be solubilized in a DPC/SDS mixture for solvent exchange before
reconstitution back into DPC [
39
].
One way to get around this problem is to treat the protein as a two-domain
system, since non-exchangeable sites tend to be clustered together for several
contiguous residues along the protein sequence [
250
]. As shown for the tetrameric
potassium channel protein KcsA in SDS, a complementary pair of samples can be
generated; one that retains amide protons at the solvent-exposed sites, and the other
with protons at non-exchangeable sites [
138
]. The former sample is generated using
conventional protocols for perdeuteration followed by amide-proton exchange with
