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Figure 4.
Fig. 4. Relaxation kinetics of EnHD residues 16–59
monitored by fluorescence (a, b, and f) and IR (c and d) and
equilibrium properties (e). (a) Kinetic time course for EnHD
residues 16–59 at 25°C, 2 M [NaCl]. The raw data (red)
were fitted after subtracting from the NATA trace (blue), giving
the rate constant of 206,000 s^–1 (4.8 µs). (b) Kinetic
time course for EnHD L16A at 25°C, 2 M [NaCl]. As for the
fragment, the raw data were subtracted from the NATA trace and
fitted to both single (gray) and double (brown) exponentials.
The single-exponential fit gave a rate of 44,000 s^–1, and the
double-exponential fit produced rates of 34,000 s^–1 and
205,000 s^–1. (c) Folding of EnHD L16A (red) and 16–59
fragment (blue), monitored by IR at 25°C, 100 mM [NaCl]. (d)
As in c, but measured at 500 mM [NaCl]. Two kinetic phases were
observed in the folding of L16A and 16–59 fragment at low salt
concentrations when monitored by IR: the fast phase and the
(previously unidentified) ultrafast phase. In addition to these
phases, a slow phase was observed in the folding kinetics of
L16A at 500 mM. No slow phase was observed in the folding
kinetics of the fragment at high salt, as shown in Table 1. (e)
Thermal denaturation of the fragment at increasing salt
concentrations, followed by CD at 222 nm. (f) Rates extracted
from independent traces at multiple temperatures for the
fragment (red) and the L16A mutant (blue/gray) at 2 M [NaCl].
The spread in the data shows the inaccuracy of the measurement
and/or data fitting. The L16A data were fitted to a single and a
double exponential. At higher temperatures, it was not possible
to fit the L16A data to a double exponential.
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