Biomedical Engineering Reference
In-Depth Information
in a spectrum which is less reliable than their appearance. Using e.g., 50% U-
[
13
C]- and 50% U-[
12
C,
15
N]-labelled protein, allows direct detection of
intermolecular
13
C-
15
N contacts.
57
A further extension of this concept uses a
mixture of protein labelled using [1-
13
C]- and [2-
13
C]-glucose.
58
Due to the
characteristic labelling patterns obtained when using [1-
13
C]- or [2-
13
C]-glucose
in the bacterial growth medium certain cross-peaks can be unambiguously
identified as inter- rather than intramolecular cross-peaks.
The use of deuterated protein has also proved useful in many circumstances
by improving linewidths
48
or enabling proton-detection experiments.
59
13.5 Structure Determination
The microcrystalline SH3 domain of a-spectrin was the first full protein
structure to be determined by solid-state MAS NMR.
1
While previous
structure determinations of small peptides and ligands centred on the use of
highly accurate distances determined using transferred-echo double-resonance
(TEDOR) techniques, the SH3 study made use of a high number of less
accurate distance restraints obtained from spin-diffusion experiments.
1
Torsion angle restraints obtained from chemical shifts were later added to
the protocol.
60
The approach to structure determination thus moved much
closer to that used in solution NMR and has since remained broadly similar.
The repertoire of pulse sequences has, however, dispensed with proton-driven
spin-diffusion (PDSD) experiments,
61
which have largely been superseded by
dipolar-assisted rotational resonance (DARR) experiments
62
to obtain
13
C-
13
Cor
15
N-
15
N distance restraints.
1
H-
1
H distances can be probed
indirectly using CHHC and NHHC experiments.
63
More recently third-spin-
assisted
64,65
and radiofrequency dipolar recoupling (RFDR)
66
pulse sequences
have been used to obtain a large number of long-range distance correlations
simultaneously.
A further feature of the SH3 structure determination was the use of the
[1,3-
13
C]- and [2-
13
C]-glycerol labelling scheme. The resulting checkerboard
labelling improved linewidths (by removing scalar couplings) and reduced
spectral crowding (by reducing the number of
13
C-labelled sites in the protein).
Crucially, the labelling scheme also increased the number of long-range
correlations that could be observed by removing many large dipolar couplings
between directly bonded
13
C nuclei that tend to dominate the flow of
magnetisation pathways. Although a more recent structure determination of
ubiquitin showed that sufficient long-range distance restraints for a full
structure determination can be obtained using uniformly labelled protein,
67
the
extensive labelling schemes such as that based on [1,3-
13
C]- and [2-
13
C]-glycerol
remain popular.
Much of the method development for structure determinations in the solid
state have been conducted on microcrystalline model proteins. A very high-
resolution structure of GB1, for instance, was obtained when including vector
angle restraints derived from HN and HC dipolar couplings.
53
Many methods
