Biomedical Engineering Reference
In-Depth Information
potential to observe correlations between C
a
and side-chain carbonyl carbons
as well as aromatic carbons. This requires low-power broadband mixing
schemes, which are under development in our laboratory.
A 3D version of this experiment, which avoids signal overlap in larger
proteins, might also be of interest. Although the sensitivity of the experiment
was largely improved by using the mononuclear TOCSY transfer step rather
than C-N heteronuclear coherences, further improvement in sensitivity might
be needed for routine use of this experiment for very large single-polypeptide-
chain proteins. In this regard, an experiment utilising the larger
1
H
polarisation as a magnetisation source but not for detection can be
considered.
43,55
In our experiments, 3D hNCACA-TOCSY 3D had almost
the same sensitivity as CACA-TOCSY despite the longer magnetisation
transfer pathway (Takeuchi et al., to be published). In addition, a proton-
excided/detected 3D experiment that contains a CACA-TOCSY transfer block
would provide unique sequential information and was published recently.
75
2.5
15
N
H
-Detection Experiments for Main-Chain
Resonance Assignments
In a rough estimate, the intensity of a signal is proportional to the c of the
excited nucleus and to c
3/2
of the detected nucleus. Thus, assuming equal
polarisation and no relaxation, the signal intensities in experiments detecting
15
N nuclei are expected to be four times lower than when detecting
13
C.
However, as discussed above,
13
C-direct detection in a uniformly
13
C-labelled
protein is not straightforward, especially for
13
C
a
-direct detection. The
13
C-
12
C alternate labelling strategy is simple and has high sensitivity for
many sites. However, there is a complicating variation of the
13
C
a
-labelling
probability.
50
On the other hand,
15
N exhibits no homonuclear coupling as it is
only attached to proton and carbon(s). Removing one-bond J couplings in a
15
N direct-detection experiment is rather easy even in uniformly
13
C-labelled
samples. In addition, the sensitivity losses due to the lower c of
15
N nuclei can
at least partially be compensated by the slower transverse relaxation of
15
N.
Two
15
N direct-detection triple-resonance experiments, CAN and CON,
have been presented to date.
41
The CAN experiment provides sequential
connections between
15
N
H
resonances using
13
C
a
chemical shift matching
[Figure 2.9(A)]. In principle, the CAN nitrogen-detected experiment provides
the same correlations as the
13
C-detected NCA experiment.
27,50
In practice, it
may provide additional resolution in the nitrogen dimension and it can be
achieved with a simpler pulse sequence resulting in a comparable but more
uniform sensitivity. The CAN experiment can be complemented with a
15
N-
detected CON experiment, which correlates the amide nitrogens of residue i,
15
N
Hi
, to the
13
C9 spin in the proceeding residue (
13
C9
i21
˜
The combination of
the two experiments yields assignment of backbone heavy atoms (
15
N
H
,
13
C
a
,
13
C9) including prolines.
