Chemistry Reference
In-Depth Information
data required to determine their structures. We will outline current approaches
being used to address these challenges and review the progress being made to
increase the range of membrane protein systems that can be studied by solution
NMR. It should be noted that solid-state NMR also continues to make impressive
advances in the study of membrane protein structure and dynamics, as interested
readers can refer to in these authoritative reviews [
16
-
19
].
2 Production of Membrane Protein Samples for Solution NMR
2.1 Factors Affecting Choice of Expression System
Solution NMR of membrane proteins usually requires that uniformly
15
N,
13
C-labeled samples be produced to facilitate chemical shift and NOE assignments.
Samples with uniform or site-specific incorporation of
2
H atoms are also often
needed to reduce the number of unfavorable relaxation pathways that can signifi-
cantly attenuate the NMR signal in large protein-detergent complexes [
20
].
Although the price of these isotopes has come down over the last decade, cost-
effective production of multiple NMR samples still requires the use of expression
systems that can produce a high yield of the target protein from the simplest
possible metabolic precursors.
Escherichia coli
is widely regarded as an ideal
expression host for this purpose since it is simple to use, can produce high levels
of protein, and has available a large variety of expression plasmids and strains for
this purpose [
21
-
23
]. In addition, condensed phase approaches that allow large
volumes of bacterial culture in unlabeled media to be resuspended in reduced
volumes of labeled media for protein expression can further reduce associated
costs [
24
-
28
]. Although yeast and mammalian cell expression systems are also
being developed as alternate sources of isotope-labeled protein [
29
-
33
], the conve-
nience of the bacterial expression system has preserved its dominance as a host for
NMR sample production. Notably, almost all membrane protein structures that
have been determined by solution NMR to date were produced using proteins
expressed in
E. coli
, or peptides produced through solid-phase chemical synthesis
(nicely summarized at
http://www.drorlist.com/nmr/MPNMR.html
).
2.2 Membrane Protein Expression in
E. Coli
Special considerations for membrane protein expression in
E. coli
include issues of
targeting; ideally the expressed protein can be incorporated into the bacterial cell
membrane, allowing extraction of folded samples from detergent-solubilized cell
membranes [
34
-
37
]. This has been the case for the small number of polytopic
helical membrane protein structures that were successfully determined by solution
NMR, namely diacylglycerol kinase (DAGK) [
38
], the disulfide bond isomerase
DsbB [
39
], sensory rhodopsin II (pSRII) [
40
], and the mitochondrial uncoupling
