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Figure 8.
Typical
15
N relaxation dispersion profiles for KIX displaying residues Glu593 and Val635, recorded at static
magnetic field strengths of 11.7T (red) and 18.8T (blue) at 26.9
o
C and in the presence of 0.4M
myo
-Inositol and 0.4M
pinitol. Error bars represent uncertainties in relaxation rates. CPMG radio frequency field strengths, v
CPMG
, ranged from
40 to 960 Hz, relaxation delays were 50ms. Spectra were collected as series of two-dimensional data sets. Duplicate
data sets were recorded at selected v
CPMG
values for error analysis. Peak intensities observed from
1
H-
15
N spectra were
converted into effective relaxation rates (R
2.eff
) and uncertainties in relaxation rates were calculated from repeat ex‐
periments. R
2.eff
were calculated by numerical modeling of magnetization evolution during the CPMG sequences. Fit
curves were obtained by combining the dispersion of all residues in a collective fit to a two-state process
15
N single quantum relaxation dispersion experiments were performed to characterize alter‐
ations in the two-site conformational exchange of the KID-binding (KIX) domain of CREB-
binding protein (CBP) in the presence of osmolytes under native conditions. Conformational
exchange of KIX
15
N backbone resonances has been shown to be in the intermediate to slow
time regime. CPMG-type relaxation dispersion data showed that under non-denaturing con‐
ditions, KIX permanently exchanges between its folded (native) ground state (G) and a par‐
tially unfolded high-energy state (E) that is populated to 3±0.2% at 26.9
o
C and pH 5.5.
Relaxation dispersion experiments were performed for KIX and in the presence of 0.4M os‐
molytes (pinitol,
myo
-Inositol, quebrachitol, quercitol), operating at static magnetic field
strengths of 11.7 and 18.8T at 26.9
o
C.
15
N relaxation dispersion profiles were fit for each site
individually (G↔E) to yield site-specific values of G→E and E→G rate constants (k
GE
and
k
EG
) and differences in resonance frequencies between G and E states │Δω
fit
│. Dispersion
profiles of all sites were then fit to a global two-site model assuming uniform values for k
GE
(k
u
) and k
EG
(k
f
), but specific values for │Δω
fit
│ (Table1). Dispersion profiles (R
2.eff
/V
CPMG
)
are dependent on k
f
and k
u
rate constants or the population of the unfolded state p
E
and the
exchange rate constant (k
ex
= k
f
+ k
u
) and on chemical shift differences between the folded
and unfolded state │Δω│[53].
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