Chemistry Reference
In-Depth Information
yielding spectra of comparable quality to those obtained in the inactivating DPC
conditions used to solve its structure [
148
]. A potential explanation for this arose
from the observation of NOEs between the glycerol spacer of lyso-myristoyl
phosphatidylcholine (LMPC) and Trp indole side chains at the peripheral regions
of the TM helices; in DPC these same side chains showed NOEs to the
phosphocholine headgroup. Based on these observations it was suggested that
these indole-phosphocholine interactions were destabilizing, and that the glycerol
spacer provided a more appropriate environment for interfacial side chains. The
potential utility of detergents with polar spacers to improve the stability of mem-
brane proteins has also been highlighted by synthetic variants of DPC with
modifications that mimic the properties of the polar spacer [
151
]. In this study
superior stabilization of OmpX for folding and solution NMR was obtained for
DPC with a
-hydroxylated, or ethyl-amide-linked alkyl chain.
Lysolipids have also proven useful for direct resolubilization of membrane
proteins from cell-free expression pellets for solution NMR structure determina-
tion, as was illustrated for three bacterial membrane proteins [
88
]. However, these
structures were all characterized by loose helical packing, which may reflect a
destabilizing influence of LMPG on these proteins. Alternatively, the loose
structures could have been a consequence of the type and number of structural
restraints used, leaving unanswered the question of possible denaturing effects of
this detergent on these structures.
b
3.1.5 Mixed Micelles
For some proteins an appropriate balance between solubilization and stabilization
could not be provided by a single detergent system, but was attained using a mixture
of detergents. For example, the TM helix
dimer could only be solubilized in SDS
after its final purification step, but could subsequently be transferred into a less
denaturing system by the addition of a fivefold molar excess of DPC over SDS, with
samples undergoing aggregation if the ratio of DPC:SDS exceeded ~10:1 [
152
].
Similarly, NMR spectra of the KvAP voltage sensing domain were found to be
optimal in 2:1 DPC/LDAO (lauryldimethylamine-oxide), with DPC alone giving
rise to exchange-broadened spectra, while LDAO yielded favorable spectral
properties but short sample lifetimes [
153
]. An N-terminal fragment from the Y4
GPCR containing two TM segments also benefited from the use of a detergent
mixture (i.e., DPC and lyso-palmitoyl phosphatidylglycerol (LPPG)), since DPC
could solubilize the sample but yielded spectra with broad lineshapes, while LPPG
was a poor solubilization agent for this sample [
154
]. The common theme arising
from these studies is that detergent mixtures can offer an improved capacity to
provide the best compromise between sample solubility and stability. Also, in some
cases it may be necessary to include small amounts of phospholipids normally
found in the membrane environment, as was required for the mitochondrial
uncoupling protein 2 (UCP2) [
41
].
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