Figure 5.
FIGURE 5. Thermodynamics and kinetics of the BAF[2]-Em^LEM
complex. A, ITC titration of BAF[2] with Em^LEM. The titration
(3 µl per injection of 854 µM Em^LEM) was performed
at 30 °C in a calorimetric cell ( 1.8 ml) containing 31
µM BAF[2] dimer in 25 mM Tris-HCl, pH 6.5, and 0.2 M NaCl.
The experimental data are shown as solid circles. The best-fit
curve to a one site binding equilibrium is shown as a solid line
and yields a value of K[d] = 0.59 ± 0.03 µM. B,
because one molecule of Em^LEM binds to the BAF[2] dimer, the
chemical environments of equivalent residues from the two
subunits of BAF are no longer identical and display different
chemical shifts, as illustrated for Gly^47. Z-exchange
spectroscopy reveals the presence of exchange cross-peaks
(indicated by ex) between equivalent residues in addition to the
auto-peaks (labeled as G47 and g47'). This arises from the fact
that Em^LEM can bind to the BAF[2] dimer in two chemically
equivalent ways related by a 180° rotation (see Fig. 6). C,
kinetic scheme describing the magnetization transfer involving
dissociation and reassociation of Em^LEM to BAF[2] in two
chemically equivalent orientations. Cross-peaks corresponding to
the two magnetically inequivalent subunits of BAF[2] in the
complex are simply interchanged in the two bound states. M[F] is
the magnetization of free BAF[2]; M[B] and M[B]['] are the
magnetizations of the two bound states of BAF[2] related by the
180° rotation of Em^LEM; k[on] and k[off] are the
association and dissociation rate constants, and [Em^LEM][F] is
the concentration of free Em^LEM; [F] and [B] are
the spin-lattice relaxation rates for free and bound BAF[2] and
for simplicity are considered equal because [F] cannot be determined
from the present data. D, time course of the normalized
auto-(open circles) and exchange-(closed circles) peaks of
Gly^47 together with the best-fit curves (red and blue lines,
respectively) obtained for the kinetic model shown in C. The
experimental data are shown at three different concentrations of
free Em^LEM (0.39, 0.68, and 0.89 mM) with total concentrations
of U-^15N/^13C/^2H/[methyl-^1H]Val/Leu/Ile-labeled of 0.42,
0.35, and 0.30 mM, respectively, and total concentration of
unlabeled Em^LEM of 0.81, 1.03, and 1.19 mM, respectively. E,
selected strips from a three-dimensional
^12C-filtered/^13C-separated NOE spectrum illustrating
intermolecular NOEs from ^12C-attached protons of the BAF[2]
dimer (F[1]dimension) to ^13C-attached protons of Em^LEM (F[3]
dimension). The spectrum was recorded in 95% H[2]O, 5% D[2]O.
The cross-peaks involving equivalent residues in the two
subunits of BAF[2] are indicated by upper and lowercase
one-letter codes.
The above figure is reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
14525-14535)
copyright 2007.
1.8 ml) containing 31
µM BAF[2] dimer in 25 mM Tris-HCl, pH 6.5, and 0.2 M NaCl.
The experimental data are shown as solid circles. The best-fit
curve to a one site binding equilibrium is shown as a solid line
and yields a value of K[d] = 0.59 ± 0.03 µM. B,
because one molecule of Em^LEM binds to the BAF[2] dimer, the
chemical environments of equivalent residues from the two
subunits of BAF are no longer identical and display different
chemical shifts, as illustrated for Gly^47. Z-exchange
spectroscopy reveals the presence of exchange cross-peaks
(indicated by ex) between equivalent residues in addition to the
auto-peaks (labeled as G47 and g47'). This arises from the fact
that Em^LEM can bind to the BAF[2] dimer in two chemically
equivalent ways related by a 180° rotation (see Fig. 6). C,
kinetic scheme describing the magnetization transfer involving
dissociation and reassociation of Em^LEM to BAF[2] in two
chemically equivalent orientations. Cross-peaks corresponding to
the two magnetically inequivalent subunits of BAF[2] in the
complex are simply interchanged in the two bound states. M[F] is
the magnetization of free BAF[2]; M[B] and M[B]['] are the
magnetizations of the two bound states of BAF[2] related by the
180° rotation of Em^LEM; k[on] and k[off] are the
association and dissociation rate constants, and [Em^LEM][F] is
the concentration of free Em^LEM; 
[F] and