Biomedical Engineering Reference
In-Depth Information
Compared to the NCA double-IPAP (NCA-DIPAP) experiment, the carbon
transverse period of the CAN experiment is significantly shorter (28 ms in
CAN compared to 44 ms in NCA-DIPAP). Instead, the CAN experiment
spends 22 ms on a slower relaxing nitrogen-transverse period. Thus, in the
CAN experiment, signal losses due to relaxation of coherences during the pulse
sequence are significantly less than in the NCA-DIPAP experiment. This
ameliorates the intrinsic low sensitivity of the CAN experiment. For
comparison, both the CAN and the NCA-DIPAP experiments were recorded
on a 4 mM sample of the B1 domain of protein G (GB1) uniformly
2
H,
15
N,
13
C-labelled and dissolved in D
2
O buffer, supplemented with 3 mM Gd
(DTPA-BMA). It is worth noting that it becomes possible to accumulate scans
y3.3 times as fast in the presence of the relaxation agent, which in turn
represents a y1.8 fold gain in S/N.
Figures 2.9(B) and (C) show the nitrogen-detected 2D CAN spectrum and
the carbon-detected 2D NCA-DIPAP spectrum. Comparison of slices for the
same correlation demonstrates that the linewidth in the direct dimension is
much narrower in CAN than in NCA. Whereas the linewidth (LW) for
nitrogen is almost comparable to the value estimated from T
2
, the LW for
carbon was much broader than the estimated value. This emphasises the
difficulty of achieving a perfect decoupling in the DIPAP scheme. In addition,
the application of selective C
a
pulses in the DIPAP scheme leads to signal loss
in several residues.
In total, the median of the S/N in the CAN experiment is 16% higher than
that of the NCA-DIPAP experiment. The average of the S/N was even higher
for the nitrogen-detected experiment since Gly, Ser and some high-field C
a
signals are significantly weaker in the NCA-DIPAP experiment. Thus, it seems
that the lower c of
15
N nuclei was fully compensated by the slower
15
N
transverse relaxation rate, a better decoupling scheme for the detected nuclei,
as well as the relaxation-optimised properties of the pulse sequence.
Although, the spectra shown in Figure 2.9 were recorded in D
2
Oto
minimise
15
N and
13
C
a
transverse relaxation rate at this magnetic strength
(11.7 T), one can also consider an experiment that runs in H
2
O and detects
nitrogen coherences without proton decoupling. It would be particularly
interesting to test this type of experiment in a high magnetic field, as the
TROSY component of the nitrogen signal can be detected as very narrow lines.
As shown in Figure 2.1, it is predicted that the transverse relaxation of
15
N
coupled with deuterium is slowest in lower fields (11.7 T) but the TROSY
component of
15
N coupled with H is the narrowest in higher fields (18.8 T).
The 2D CAN experiment was also recorded for the 52 kDa GST protein
dimer. The T
2
values of GST were shorter than expected most likely due to the
presence of partial sample aggregation. Nevertheless, y250 resolved signals
were observed in a 3.5 day experiment, which correspond to y54% of the total
expected resonances [Figure 2.10(A)]. The observed resonances are reasonably
narrow and well dispersed, indicating the applicability of the CAN experiment
to higher molecular weight protein systems. The nitrogen-detected experiments
