Biomedical Engineering Reference
In-Depth Information
The CACA-TOCSY experiment with alternate
13
C-
12
C labelling. (A)
Pulse scheme of the CACA-TOCSY experiment optimized for uniformly
2
H
15
N- and alternate
13
C-
12
C-labelled samples. (B) CACA-TOCSY
spectra recorded at various mixing times (13, 132 and 251 ms) with
[U-
2
H,
15
N + 2-
13
C alt]-GB1 (5 mM) in 20% glycerol at 285 K. As easily
analysable areas, the regions containing correlations to glycine residues
(dotted boxes) are enlarged on top of each spectrum. Black and cyan
arrow heads in the enlarged section at 251 ms mixing time indicate
sequential and long-range (i:i 2 2ori:i + 2) correlations between C
a
nuclei, respectively. The measuring time for each spectrum was 5.5 h. (C)
Examples of build-up curves for three sequential C
a
i
-C
a
i+1
correlations
(residues 49-50, 50-51, and 51-52). (D) Location of residues that have
fast and slow build-up rates, respectively. On the 3D structure of GB1,
C
a
i
-C
a
i+1
correlations with ratios of intensities between 132 ms and 251
ms, I132/I256 $1.5 or #1.0 are indicated by the red and cyan spheres in
C
a
i
positions, respectively. Details of the experiment can be found in the
original literature. Adapted from ref. 71.
Figure 2.8
which exclusively relies on hetero nuclear
1
J couplings. As reported previously,
the 2D CACA-TOCSY, on average, has S/N 3.7 times higher than that of the
2D CA(N)CA experiment.
71
Exceptions are the C
a
-C
a
correlations involved in
the residues that are
13
C-labelled simultaneously in the C
a
and C
b
positions
(i.e., Val and Ile with 2-
13
C glycerol). For these residues the strong
1
J carbon-
carbon coupling causes detrimental coherence 'leaking', which prevents the
weak C
a
i
-C
a
i+1
correlations to be observed.
The CACA-TOCSY experiment with alternate labelling also correlates C
a
to
specific side-chain carbons in certain types of amino acid residues. As reported
before,
71
the information can be quite useful for identifying the amino acids
type. Val (C
b
), Ile (C
b
), Met (C
c
), Thr (C
c
), Arg (C
d
), and Lys (C
d
) can be
identified in 2-
13
C-labelled samples and Arg (C
c
), Glx (C
c
), Pro (C
c
), and Leu
(C
d
)
1,3-
13
C-labelled
in
samples.
The
experimental
scheme
also
has
the
