Biomedical Engineering Reference
In-Depth Information
Fig. 16.4
Residual dipolar couplings measurement and membrane proteins aligned in presence of
the six-helix bundles. (
a
) Superposition of the expanded region of
1
H-
15
NHSQC(
blue peaks
)
and TROSY (
red peaks
) spectra for the isotropic and anisotropic sample. The difference in
15
N frequency between the
blue
and
red
peaks is either
1
J
NH/2 for the isotropic sample or
(
1
J
NH
1
D
NH)/2 for the aligned sample.
1
D
NH can be determined from the two values. The
15
N-
1
HN splittings (in hertz) are marked together with the sequential assignment. Spectra recorded
at 25
ı
Cat
1
H frequency of 600 MHz. The sample condition is 500
C
M uniformly
15
N-labeled
ubiquitin in 20 mM tris and 200 mM NaCl, pH 7.5. (
b
) Correlation between measured
1
H-
15
N
RDCs of ubiquitin in 20 mg/mL DNA nanotubes and couplings calculated on the basis of X-ray
crystal structure of ubiquitin pdb code: 1UBQ. Linear regression applied to the data gives a cor-
relation coefficient of 0.993. Structure of ubiquitin (pdb code: 1UBQ) shown with the orientation
of the principal components of the alignment tensors in grey. (
c
) NMR structures of '-helical
membrane proteins aligned in presence of the six-helix bundles. Structures of ——
TM
domain in
dodecylphosphorylcholine and dodecylsulfate, DAP12
TM
, both alone and covalently linked with a
receptor transmembrane domain NKG2C-DAP12
TM
trimeric transmembrane peptide complex in
mixed tetradecylphosphorylcholine, dodecylsulfate detergent micelles, BM2
TM
in (1-myristoyl-2-
hydroxy-sn-glycero-3-[phospho-rao-(1-glycerol)]), and UCP2 in DPC and cardiolipin. All models
were produced using the PDB files (Color figure online)
16.5.6
RDC Calculations
RDC calculations are done by subtracting the values of splittings measured in
unaligned (
J
) and aligned (
J
C
D
) samples. The sign of the RDC follows the con-
vention that
j
92 Hz when
1
D
NH is positive. In Fig.
16.4
b,
1
H-
15
N
RDCs of the ubiquitin protein were obtained from
1
J
NH/2 and (
1
J
NH
C
1
J
NH
C
1
D
NH
j
<
1
D
NH)/2,
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