Chemistry Reference
In-Depth Information
protein G (OmpG) [
100
,
101
]. A selective labeling scheme has also been used in the
HEK293 system to express labeled rhodopsin for structure and function study
[
12
,
18
,
22
,
102
-
104
]. The reverse labeling approach has been designed to study
structure and dynamics of the sensory rhodopsin II and the potassium ion channel
KcsA-Kv1.3 [
105
,
106
]. This approach has the advantage of decreased numbers of
overlapping resonances from the hydrophobic transmembrane helices, making the
extensive assignments possible. By taking advantage of the glycolysis pathway, the
TEASE
13
C selective labeling scheme has been suggested to probe the transmem-
brane segments of membrane proteins [
107
], and this reverse labeling approach
may possibly be adaptable for other different applications for its flexibility.
A uniformly labeling scheme is the simplest and most cost-effective biosynthetic
labeling method for protein SSNMR. Normally, uniformly
13
C-labeled glucose or
glycerol, and
15
N-labeled ammonium chloride or ammonium sulfate are used as the
labeled precursors in the growth medium. With a single sample, all the structural
constraints can be obtained through a set of correlation experiments and the protein
structure can be calculated thereby. This approach has been first demonstrated on
microcrystalline proteins of known structure [
108
-
111
], and then successfully
applied to membrane protein for structure determination [
7
,
38
,
41
,
105
,
112
-
114
].
Recently, predeuteration of proteins has been exploited to gain even higher
resolution for U-
13
C and
15
N labeled proteins in SSNMR by taking advantage of
minimizing the
1
H-
1
H dipolar couplings causing spectral broadening. Perdeuterated
soluble microcrystalline proteins have been used to study protein dynamics and
interactions [
115
-
117
], and structure determination [
118
]. This approach has also
been attempted to express the fully labeled 7-transmembrane protein bacteriorho-
dopsin for structure investigation, as shown in Fig.
1
[
119
].
However, with increase of deuteration level, protein expression level may
drop and some strains may even be difficult to grow on D
2
O[
120
]. Increasing
deuteration level may also influence the resonance frequencies and CP efficiency.
Therefore, a good balance needs to be considered from both the NMR and
biological sides.
Choosing an appropriate sample environment is not only critical for generating a
high resolution NMR spectrum, but also critical for the protein to have correct
folding and activity. Obtaining homogeneous sample preparation leads to improved
linewidths and therefore spectral resolution, while heterogeneous samples can
result in artifacts such as unexpected peaks and peak doubling [
120
]. Furthermore,
it is not enough to show that a protein construct is functional to validate a structure
unless the functional assay is performed in the same environment as that used for
the structural characterization [
121
]. For small proteins, nano-/micro-crystalline or
nano-disk samples have been proven a good choice for yielding high quality spectra
in solids [
122
-
124
]. For membrane proteins, samples can be prepared in either
detergent micelles, bicelles, or lipid bilayers. Given that membrane proteins func-
tion within a bilayer environment, it is more biologically applicable to be able to
carry out structural investigations in lipids [
120
]. Structural data obtained in an
appropriate lipid bilayer environment can serve as benchmarks for validating
structures determined in other mimetic environments [
121
]. Figure
2
shows the
