Biomedical Engineering Reference
In-Depth Information
15
N
D
in the literature.
40
down to 0.7 Hz in some signals were reported for
On
15
N linewidths of $3 Hz were observed in a
15
N-detected
the other hand,
spectrum of protonated ubiquitin in H
2
O.
The proton-attached
15
N
H
lines are anisotropic as studied by the DD-CSA
interference especially in a larger molecular weight systems in high magnetic
fields.
3
The TROSY effect is field dependent and is close to optimal already in
an 18.8 T magnet.
4
As shown in Figure 2.1(A), the linewidth of the 'TROSY'
component of the
15
N
H
line becomes narrower at higher field strength, and, at
18.8 T, it is even slightly better than a deuterium-attached
15
Ninalow
magnetic field. These observations clearly indicate that it is beneficial to detect
the lower c nucleus,
15
N, especially the
15
N
H
TROSY line in large molecular
weight systems in high magnetic fields.
As expected, perdeuteration of a protein has only a marginal effect on
13
C9
and
15
N relaxations but significantly narrows the
13
C
a
resonances. Even for
perdeuterated proteins in D
2
O, the dominant contribution to the
13
C
a
relaxation is the DD interactions (y90% of relaxation source). Thus, the
13
C
a
resonances in deuterated proteins are almost independent of the magnetic
field strength and provide the best opportunity to exploit the sensitivity gain
from high magnetic fields. This is clearly seen by the fact that the transverse
relaxation rate of a deuterated
13
C
a
resonance is comparable to that of the
lower c
15
N
D
in a high field (18.8 T). The deuterated
13
C
a
resonance can also
be detected in H
2
O instead of D
2
O as the protonation in an amide position
accelerates the transverse relaxation of
13
C
a
only by y15%.
Direct
13
C
a
-detection experiments, however, are complicated due to the
scalar couplings with both C9 and C
b
causing crowded spectra and reducing
sensitivity due to splitting peaks into multiplets. However, as we will discuss
below, the spectral complexity can be avoided by selecting a single component
within the split peaks,
49
or by the recently introduced alternate
13
C-
12
C-
labelling scheme.
50-52
These techniques will be discussed more in detail in later
sections.
Since the longitudinal relaxation times (T
1
) of low-c nuclei can be long in
large-molecular-weight systems, the repetition delay has to be carefully set to
have optimal sensitivity. The sensitivity of the experiment can be further
enhanced by shortening T
1
using paramagnetic reagents, such as Gd(DTPA-
BMA) or Ni(DO2A) without significantly accelerating transverse relaxation
rates.
28,53
Indeed, it is reported that the addition of 3 mM Gd(DTPA-BMA)
decreased the T
1
values of deuterated
13
C
a
from 3.6 to 0.9 s.
51
This allowed
faster recycling rates and the acquisition of more scans per increment and
increased the sensitivity of the experiment by y50%. The paramagnetic
relaxation enhancement can be less effective for the inaccessible core of very
large proteins. We observed, however, that the average
13
C
a
T
1
values of the 52
kDa dimeric protein GST were also significantly shortened throughout the
protein from approximately 3.6 to 1.3 s, by addition of 4mM Gd (DTPA-
BMA).
51
