Chemistry Reference
In-Depth Information
with the membrane normal being parallel to the field [
79
,
80
]. The binding of the
GDP-bound form of the heterotrimeric G protein transducin (Gt) to the light
activated MII intermediate of the rhodopsin activates the visual signal transduction
pathway. A peptide consisting of the C terminal ten residues of the
a
-subunit of
a
transducin (Gt
) is capable of competing with Gt for binding and was studied using
NMR spectroscopy [
81
]. In the presence of light, Gt
a
transiently binds activated
GPCR that is embedded in membrane disks of bovine retina. Since these disks were
aligned in the magnetic field, RDCs for Gt
could be observed. The measured
RDCs followed a time-decay course after light activation, while GPCR returned to
its inactive state. The decay time constant is on the order of 1 h that allowed rapid
acquisition of 2D
1
H-
15
N and
1
H-
13
C spectra on the Gt
a
peptide sample. The RDC
values at zero time were obtained from extrapolation of multiple RDCs collected
along a time decay curve. The structure and orientation of the peptide in the bound
state to GPCR were derived from RDCs and transferred NOEs [
81
]. The validity of
such use of RDCs relies on two conditions. The first condition is the fast exchange
between the free and bound states of Gt
a
that allows the observation of only one set
of resonances of the free state. The other condition is that the free Gt
a
peptides are
not aligned in GPCR enriched PM media so that alignment contribution all comes
from the bound state. Indeed the alignment order of the system was weak with a
D
a
value of only 1.6 Hz.
In the above case, the ligand receptor GPCR is naturally buried in bicelles, which
by itself is the alignment medium. This ensures strong alignment order for bound
ligand molecules. However, for a general study protein receptors are not necessarily
buried in any disks that could be aligned; thus the alignment order for ligand
molecules in equilibrium with its free form would be too weak to be observed.
Seidel et al. [
82
] demonstrated a method of anchoring polyhistidine-tagged protein
receptors onto the bicelles that were doped with histidine-tag binding lipid
molecules. This increased the alignment order of the protein-ligand complex and
prevented measurements of the protein RDCs. The RDCs of the bound ligand in fast
exchange with free ligand molecules, however, were observed and they could be
used to map the ligand configuration on the protein receptors.
a
4.5 Structure of Oligomeric State
RDCs are very useful in improving the structure determination of a monomeric
protein. In addition, they can be utilized to establish quaternary structure of
symmetric oligomers. Prestegard and coworkers have developed methods to deter-
mine dimer structure using RDC data [
83
,
84
]. The underlying principle is that the
rotational symmetric
C
2
axis of any protein dimer should be parallel to one axis of
the alignment tensor of the dimer. By solving alignment tensor axes from different
alignment media, one could identify the common tensor axis that should be parallel
to the symmetric axis [
85
,
86
].
Bacillus subtilis
proteins YkuJ dimerize with strong
affinity and its
K
d
is on the order of 10
9
M[
83
]. When working at the typical NMR
