Chemistry Reference
In-Depth Information
where the recoupling is achieved through rotational resonance, or by rf pulse driven
methods, where the recoupling is achieved by applying rf pulse trains. By applying
recoupling sequences, certain anisotropic interaction terms can be retrieved in the
presence of MAS. Magic-angle spinning NMR in combination with recoupling has
become a widely used experimental technique for obtaining molecular structural
information in non-crystalline or disordered materials. MAS makes it possible to
obtain SSNMR spectra with good resolution and sensitivity, while the recoupling
techniques cause a selective restoration of informative anisotropic nuclear spin
interactions, which are normally suppressed by magic angle rotation. A wide range
of recoupling methods has been developed for homonuclear and heteronuclear spin
systems [
85
-
88
]. With a combination of recoupling sequences and multidimen-
sional experiment schemes, membrane protein structure and dynamic conformation
upon activation can be revealed through the short and long range semi-quantitative
distance restraints from multidimensional homonuclear/heteronuclear correlation
experiments, such as PDSD/DARR, PAR, TEDOR, PAIN-CP, and CHHC/NHHC
type experiments [
18
,
89
-
92
,
127
,
191
-
192
], etc.
3 Protein Structure Determination by MAS Solid-State NMR
3.1 Labeling Strategy and Sample Environments
Isotopic labeling plays a very important role in molecular structure determination in
SSNMR. It not only enhances the spectral sensitivity and improves the spectral
resolution, but also helps with the resonance assignments of NMR spectra and
tackles the specific problem with the structure and dynamics of proteins through the
designed labeling scheme.
There are three main approaches to isotopically label proteins: specific, selec-
tive, and uniform labeling. All are used extensively in SSNMR studies of protein
structure and ligand conformation, depending on the questions that the designed
experiment needs to be answered.
Specific labeling normally refers to incorporate a non-uniformly
13
C,
15
N
labeled amino acid into a polypeptide/protein or a ligand at a certain position. It
requires solid-phase peptide synthesis or chemical synthesis. This approach has
been used extensively to study amyloid peptides, membrane peptides, and GPCR
ligand conformation [
93
-
99
].
Selective labeling, including forward and reverse labeling, refers to the biosyn-
thetic incorporation of a single type or several types of labeled amino acid(s) - with
the rest unlabeled - into the protein expression media. Selective labeling is widely
used for the protein resonance assignments and structure calculations in SSNMR.
For example, a selectively and extensively labeling scheme using [1, 3-
13
C] and
[2-
13
C]-glycerol as the sole carbon source in the bacterial growth medium has been
used to determine the
a
-spectrin SH3 domain,
a
B-crystalline, and outer membrane
