Chemistry Reference
In-Depth Information
amide protons and not perturb water protons [
51
]. In this way, saturation of water
proton magnetization, which reduces amide proton intensity by water-amide
exchange at high-pH, is avoided. However, in practice this can be achieved only
at high fields in which there is a sufficient chemical shift separation between amide
and water proton chemical shifts. Otherwise, complete selective inversion is
achieved at the cost of putting amide proton magnetization in the transverse plane
for significant periods of time.
2.3 Practical Aspects in
15
N-{
1
H} NOE Experiment
15
N-{
1
H} NOE equals the ratio of steady state
15
N signal intensities recorded with/
without
1
H saturation. Since the
15
N signal intensities have to be accurately
encoded in the
t
1
dimension, INEPT transfer from
1
Hto
15
N is not used prior to
the
t
1
evolution. As a result, the sensitivity of the
15
N-{
1
H} NOE experiment is ca.
ten times lower than that of
15
N
R
1
and
R
2
. To compensate for the low signal-
to-noise ratio, experiment recording times are larger in the NOE experiment than
in
R
1
and
R
2
. In addition, a long magnetization recovery time (
3s
1
) between
each scan adds to the total time required to obtain data with adequate sensitivity
[
6
,
52
-
54
].
Sufficient magnetization recovery of
15
N (in the experiment with
1
Hsaturation)or
both
1
Hand
15
N (in the experiment without
>
1
H saturation) is crucial to determine
NOE values accurately. In proteins where
t
R
o >>
1, dipolar longitudinal relaxation
rates decrease as the resonance frequency,
, increases. Therefore, a longer recovery
time will be required for the
15
N-{
1
H} NOE experiment at higher magnetic field
strength. In a rigid protein with a rotational correlation time,
o
t
R
,of10ns,
15
N
R
1
is
1.1-1.4 s
1
at 61 MHz (in a 600 MHz NMR instrument) whereas
15
N
R
1
is ca.
0.69-0.85 s
1
at 91 MHz (in a 900 MHz instrument). Thus, recovery times more than
3 and 5 s are required at 61 and 91 MHz, respectively. However, these are the
recovery times estimated from
15
N
R
1
.
1
H recovery times often become longer when
there is not much surrounding
1
H nuclei or less
1
H spin-flip (such as deuterated
proteins, unfolded proteins, or in a loop region of a folded protein). In this case,
insufficient
1
H Z-magnetization recovery is corrected using the equation derived by
Bax and Grzesiek [
52
]. When the
15
N
R
1
recovery is not sufficient, another correction
equation that counts both
1
Hand
15
N
R
1
recovery is used [
55
] These corrections work
reasonably once accurate
1
Hand
15
N
R
1
values are obtained.
When the amide proton magnetization does not recover in a single exponential
manner, the correction equations do not give accurate results. In particular, when
there is severe DD/CSA cross correlation in a deuterated protein in which the
proton spin-flip rate is small at high-magnetic field strength, the decay of
1
H
magnetization of one of the two
15
N coupled components becomes slow and
nonexponential [
56
]. A simple solution will be to apply a sufficiently long recovery
time. An alternative solution will be a pulse sequence that has recently introduced
by Ferrage and coworkers [
57
,
58
].
