Chemistry Reference
In-Depth Information
tumbling becomes slower, both
15
N
R
2
and
1
H
R
1
increase (the later by proton spin-
flip), resulting in increase in
R
2
. To overcome this problem, use of
1
HCWdecoupling
scheme decrease eliminates the
1
H
R
1
term, resulting in
R
2
determined only by
15
N
R
2
[
110
]. Further reduction of
R
2
is achieved by a pulse sequence in which there is an
additional H
Z
N
Z
-relaxation period to set the
1
H
R
1
term to be time-independent, called
the “constant relaxation time” scheme [
106
,
122
] with combination of TROSY [
123
,
124
]. Alternatively, use of a deuterated
15
N protein is a simple way to reduce the
1
H
R
1
contribution in the
R
2
in
15
NCPMG
R
2
dispersion.
15
N Off-Resonance
R
1
r
Experiment
4.3
Characterization of milli- to microsecond motions in proteins based on chemical
exchange can also be performed by the dispersion version of the
15
N off-resonance
R
1
r
experiment [
39
,
106
,
125
-
128
]. As described in the section on
15
N
R
2
(Sect.
2.1
), a simple equation, (
1
), is applied to determine
R
1
r
from
R
obs
and
R
1
.
Although a strong
B
1
field strength that satisfies
o
1
>>
(
o
0
o
rot
) is applied to
minimize the
R
1
cos
2
y
term in the standard (on-resonance)
R
1
r
experiment, the
(
o
0
o
rot
) term is significant and is varied in the off-resonance
R
1
r
experiment
[
40
,
106
].
R
1
r
is plotted as a function of the off-resonance field strength,
o
e
/2
p
,
1
=
2
0
rot
given by
:
In theory,
R
1
r
is well-suited to record spectra at high
o
e
¼ðo
þ o
Þ
o
e
/2
p
values because high
o
e
/2
is achieved by increasing off-resonance field strength without increasing
B
1
field strength. However, since
R
obs
decreases as
p
o
e
/2
p
increases, a longer spin-lock
period is required to record the reduced
R
obs
and this determines the limits of
o
e
/2
p
applicable
. Although the sensitivity largely depend on sample concentration
and intrinsic
R
2
at each site, the data in the literatures cited above have been mostly
acquired for 140-200 ms with
up to 2-4 kHz.
Most of the experimental parameters are the same as those of the on-resonance
R
1
r
experiment except for a couple of points. (1) Constant relaxation-time scheme
is useful to subtract part of the
R
1
component and to simplify the
R
obs
equation
[
106
], and is also known as an “
R
1
r
o
e
/2
p
R
1
” sequence [
126
]. (2) A scheme to spin-
lock most of the magnetization uniformly is needed. For this, a scheme with periods
of evolution due to chemical shift offset [
38
,
39
,
106
] or adiabatic rotation of
magnetization by amplitude and phase modulated pulses [
125
,
127
] is applied.
When there is a large chemical shift dispersion, such as a high magnetic field
strength, the latter has been recommended [
125
]. (3) Suppression of DD/CSA cross
correlation and the artifact caused by the antiphase component is required. As
described in the section on
15
N
R
2
(Sect.
2.1
), sequences to take care of these
effects have been used, in particular to record the data at weak off-resonance field
strength [
38
,
39
]. For application to large proteins, a TROSY-selected version has
been proposed [
128
].
