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free indole ring in lipid bilayers, as indicated by an up-field shift of the C5 at the
indole ring due to the even strong hydrophobic interaction by the 1 HMAS
NOESY experiments. Currently, we are conducting further research on peptide
sequencing, composition, and insertion depth with basic and aromatic amino acid
residues in different membrane lipids.
1 H MAS NOESY experiments have been widely used in SSNMR to study
peptide-lipid interactions because of the fast axially rotation and segmental motion
of membrane lipids in the liquid crystalline phase which average out efficiently the
1 H- 1 H dipolar couplings, resulting in a high resolution 1 H spectrum of membrane
lipids under the slow MAS frequencies [ 166 ], which leads to the rapid applications
of the NOESY-type [ 167 ] of solution NMR methods to study peptide-membrane
interactions in MAS SSNMR [ 168 - 170 ].
The insertion depth of aromatic residues in membrane lipids depends not only on
the peptide sequencing and charge but also on the lipid composition, state, hydra-
tion level, and peptide/lipid molar ratio, so the insertion presents a complicated
dynamic mechanism.
3.6 Sensitivity Enhancement
SSNMR sensitivity can be enhanced by manipulating the Boltzmann factor by such
means as increasing the field strength, decreasing the system temperature, and
transferring proton polarization to a rare nucleus. However, the maximum polari-
zation transferred in the CP experiment is determined by
g S and the overall
enhancement is not big enough for the free use of multidimensional correlation
experiments on low-concentrated membrane proteins, especially for identifying the
dynamic switch of GPCRs between an inactive (R) state and an active (R*)
conformation [ 171 ]. Dynamic nuclear polarization (DNP) [ 172 , 173 ], chemically
induced nuclear polarization (CIDNP), or photo-chemically induced nuclear polar-
ization (photo-CIDNP) [ 174 - 176 ], and spin-exchange optical pumping (SEOP)
[ 177 , 178 ] have been exploited to deliver much higher spin polarizations. For
example, by transferring the polarization of electron spins to nuclei, the MAS-
DNP has been successfully applied to study the intermediate states in the
photocycle of bacteriorhodopsin, a 7TM light-driven proton pump. The enhanced
sensitivity of DNP permitted for the first time the characterization of the retinal
conformation in the K, L, and M states [ 35 , 179 , 180 ]. For detailed description of
the DNP experiment, please refer to the chapter of “Dynamic Nuclear Polarization:
New Methodology and Applications” in this topic.
The NMR signal-to-noise ratio is directly proportional to the square root of the
number of transitions and the total experimental time is mainly determined by
the repetition time between two successive transitions, that is about five times of the
spin longitudinal relaxation time T 1 . Therefore, NMR sensitivity enhancement can
be achieved by shortening the spin longitudinal relaxation time T 1 . Ishii and
colleagues have demonstrated that
g I /
1 H T 1 values of the two model proteins,
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