Biomedical Engineering Reference
In-Depth Information
pathways,
13
C
a
-labelling was more abundant with [2-
13
C] glycerol labelling
compared to [1,3-
13
C] glycerol labelling. The ten amino acid residues in Group
I will be more than 80%
13
C
a
-labelled with [2-
13
C] glycerol. For Ala, Cys, Gly,
His, Phe, Ser, Trp, Tyr and Val, almost 100%
13
C
a
-labelling is expected. On the
other hand, Group III residues would primarily be labelled with [1,3-
13
C]
glycerol. Leu in particular, cannot be labelled with [2-
13
C] glycerol. Group II
residues would be partially labelled in both labelling schemes. As the main
drawback of the
13
C-
12
C alternate labelling procedure, not all residues are
fully
13
C
a
-labelled, this may be largely compensated if
13
C
a
-labelled Leu or Glu
are added to the system. As for pyruvate, [2-
13
C] pyruvate would provide the
same
13
C
a
-labelling pattern as [2-
13
C] glycerol. [1,3-
13
C] Glycerol can be
exchanged to [3-
13
C] pyruvate as only the third position of carbon is expected
to be implemented in C
a
positions. However, in case one would also want to
detect
13
C9 resonances
same sample, [1,3-
13
C] pyruvate would be
in the
preferred.
Non-uniform
13
C
a
-labelling is not always unfavourable. If one of the C
a
s
adjacent to a
15
N
H
is not
13
C-labelled, INEPT transfer efficiency involving
15
N
H
becomes more efficient. Figure 2.2(E) shows the evolution of the intra-
residual and sequential correlations with different
13
C
a
-labelling ratios in the
neighbouring residues. Both intra-residual and sequential correlations are
transferred more efficiently with lower
13
C
a
-labelling ratios in the neighbour-
ing residues. It may be beneficial to use this strategy to enhance the sensitivity
of weak signals.
Complete deuteration at C
a
sites is critical for taking advantage of reduced
dipole relaxation. This was readily achieved by culturing E. coli in 100% D
2
O
media with protonated amino acid precursors. For several sites,
1
H
b
originating from the amino acid precursors will remain. The actual
enhancement of the C
a
transverse relaxation by these remaining protons is
estimated to be y20%. Nevertheless, perdeuteration can be achieved by
complete deuteration of amino acid precursors. We have successfully
established the strategy to inexpensively deuterated pyruvate (to be published).
It is also worth noting that removal of one-bond carbon DD interactions with
C9 and C
b
slows down the C
a
transverse relaxation by y10%.
2.4 C
a
-Detection Experiments for Main-Chain
Resonance Assignments
A 2D NCA HSQC experiment optimised for deuterated and alternately
13
C-
labelled proteins correlates in a straightforward way the chemical shiftsofC
a
nuclei with the shifts of the two neighbouring nitrogen nuclei (C
a
i
-N
i
and C
a
i
-
N
i+1
) (Figure 2.3). Thus, it allows sequential linking of the backbone nuclei.
Although the experiments reported were recorded in D
2
O to minimise the
relaxation of
15
N coherence in a low magnetic field (11.4 T), the experiment
can also be recorded in H
2
O, especially when it is recorded in a high magnetic
field. In a high magnetic field such as 17.4 T, the TROSY components of
15
N
H
