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Figure 3.
Figure 3. Analysis of T1 domain tetramer stability. a,
Fractional elution of Aplysia Kv1.1 wild type untagged
N-terminal cytoplasmic domains from immobilized tetramers,
formed by coassembly with otherwise identical His[6]-tagged
protein. Tetramers were immobilized by binding of the His[6]
squence to metal affinity resin. Elutions were performed under
constant flow using standard buffer conditions at 4 °C (blue
circle) and at 22 °C (red square). Curves are exponential decay
fits to the data, with one exponential term for 4 °C and two
exponential terms for 22 °C. The time constant is 5 h n  3
 s.e.m.
for all data points. b, Dissociation of monomers in 3 M urea.
Data were best fit with three exponentials with time constants
ranging from 2 min to 4 h at 22 °C (red square) and 9 min to 7 h
at 4 °C (blue circle). n 3
s.e.m
for all data points. c, Stability of mutant T1 tetramers as
determined by urea denaturation. The unfolding free energy of
wild type, V135R, N136A and N136D mutants are estimated to be
11.1, 10.2, 9.8 and 7.4 kcal mol-1, respectively. d, Correlation
of the energetic effects of T1 mutations on channel gating
properties with the change in unfolding free energy of the T1
domain. The functional  G
for the change in activation midpoint is proportional to the
shift in V[1/2] by the factor z[G]F, where z[G] is the gating
charge that is moved across the lipid bilayer during channel
activation gating and F is Faraday constant. The functional G
is proportional to ln (  [mutant]/
[wildtype])
by the factor -RT, for the changes in gate closing time measured
at -50 mV. Slopes and intercepts of the regression lines are:
2.2 mV kcal-1 mol-1 and -24.56 mV for the change in half
activation; and -0.18 kcal-1 mol-1 and 2.05 mV for the change in
ln ( [mutant]/
[wildtype])
measured at -50 mV.
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