Chemistry Reference
In-Depth Information
such a two-point fitting has been studied [ 117 , 118 ], it was not often applied for
the conventional relaxation experiments for proteins, which record several data
points to optimize the relaxation rate. For example, in the 15 N R 1 and R 2 experi-
ments for the model-free analysis, it must be important to ensure that the signal
decay is expressed as a single-exponential function so that the theoretical
equations are applied for the analysis. This is not the case in the analysis of R 2
dispersion because R 2 is not dissected to extract parameters for internal motion.
It will also be noteworthy that recent developments of the commercial NMR
instrument have significantly increased signal-to-noise ratio, which enables two-
point intensity measurements of 15 N R 2 of protein samples in more practical.
By using the rc-INEPT and by applying the two-point exponential fitting, the
CPMG period is held constant in the CT-CPMG relaxation dispersion experiment.
The template of this experiment was initially applied to probe side chain dynamics
of Asn and Gln NH 2 -sites, and then applied to detect backbone amide 15 N sites
[ 115 , 116 ]. There are three critical experimental parameters for the CT-CPMG
experiment: 180 CPMG pulse width, p 90 , the half duration between the CPMG
pulses,
t CP , and the total CPMG relaxation delay, T CP . To obtain the relaxation
dispersion profile, one reference spectrum without a CPMG period and a series of
spectra with a fixed CPMG period ( T CP ) but variable
t CP (a half duration between
180 CPMG pulses) are recorded. Two-point exponential fitting to determine R 2
values is done for the entire set of CPMG spectra.
R 2 values are independent of
n CP (
¼
1/(4
t CP )) when there is no chemical exchange
( R ex ¼
t CP .Incontrast, R 2 typically decreases as
n CP increases when there is chemical exchange. However, R 2 may not be independent
of
0) on the time scale similar to that of
n CP even when there is no chemical exchange, if artifacts are introduced by off-
resonance effects or CPMG pulse imperfections. To reduce these systematic errors, it
is important to apply the strongest (shortest) 180 CPMG pulses possible within probe
limits. Artifacts are maximized at 2
n (here, the n is integer) as discussed in
Sect. 2.1 [ 41 , 119 ]. Typically, the author's group employs 90
t CP f off ¼
s or a shorter 180 -
pulse at two different carrier frequencies, and records data up to 1 kHz
m
n CP .
Sample heating is a more critical issue than regular CPMG R 2 experiment
because t CP is shortened to achieve high n CP . In principle, heating of the samples
by CPMG pulses should be avoided because heating is not uniform at varying
t CP in
the CT-CPMG R 2 dispersion experiment. However, one may insert a 15 N pulse
scheme to compensate heating to perform experiments at uniform temperature
[ 120 , 121 ]. Such a compensation scheme for 1 H pulses has also been implemented
in the CT-CPMG R 2 dispersion experiment with strong 1 H CW irradiation [ 110 ].
Once the heating is so severe that the compensation sequence is required, the actual
temperatures during the experiments have to be recorded, particularly when the R 2
dispersion data are recorded at two or more static magnetic field strengths. From
this aspect, it must be better to avoid heating as much as possible.
Another critical issue in 15 NCPMG R 2 dispersion is the magnitude of R 2 in large
molecules. Since R ex is extracted from the measured R 2 , the accuracy of R ex decreases
when R 2 is large. In a pulse sequence that averages the inphase and antiphase
components, 1 H R 1 contributes to R 2 as does 15 N R 2 [ 109 , 116 ]. Thus, as the molecular
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