Chemistry Reference
In-Depth Information
of cosolvent-induced denaturation, two hypotheses have been proposed. The first
involves the cosolvent-induced promotion or destruction of the “water structure”
(Frank and Franks 1968), the second is due to cosolvent-protein binding (Timasheff
2002b). The latter presupposes a correlation between chemical denaturation (dena-
turation of protein by adding cosolvents without changing the temperature) and the
lowering of the melting temperature.
What remains to be done is to elucidate directly the mechanism upon which
cosolvents work to alter the thermal denaturation of proteins, rather than to rely
indirectly on the correlation with the chemical denaturation. Here, I apply FST to
show that it is the binding or exclusion of the cosolvents that modulates thermal
denaturation (Shimizu 2011).
The analysis can be made even simpler than the case of cosolvent-induced
denaturation described in the previous section by focusing exclusively on the
low-concentration limit of the cosolvent effect. Equation 11.2 and Equation 11.5
then simplify to
∂
∂
ln
ln
K
a
∞
∞
=
cG
(
∆∆
−
G
)
(11.10)
1
21
23
1
Tpm
,,
→
0
2
∞
∞
∆
V
=−
∆
G
(11.11)
2
21
where the Kirkwood-Buff parameter,
G
2
i
=
c
i
-1
N
2
i
, has been introduced to facilitate
the discussion.
By a combination of experimentally determined cosolvent-induced equilibrium
shifts and partial molar volume changes, one can obtain the information regarding
the interaction between the protein and the cosolvents. A systematic set of data by
Miyawaki, describing how
K
and the melting temperature
T
m
of thermal unfolding
are affected by the presence of the cosolvent molecules, will be used. The values
of ln
K
at a fixed constant temperature (333 K) for a number of cosolvents (guani-
dine hydrochloride, urea, ribose, glucose, sucrose, and trehalose) have been reported
against ln
a
1
ranging from 0.95 and 1.0 (Miyawaki 2009).
Figure 11.7 shows that Δ
G
23
is positive for denaturants, negative for stabilizers.
This suggests that more denaturants bind to the unfolded state than to the folded
state, which is in agreement with the previous discussion. The negative Δ
G
23
is attrib-
uted to the increased depletion of stabilizers when the protein denatures. Indeed,
as will be seen in the next section, the value of
G
23
for the protein's native state is
also negative for osmolytes, due to the stabilizers' exclusion from protein surfaces
(Shimizu and Matubayasi 2006). Consequently, the negative Δ
G
23
should mean that
the cosolvents are more excluded from the denatured state than from the native state.
This view is consistent with the earlier speculation derived from a molecular crowd-
ing perspective (Davis-Searles et al. 2001).
Figure 11.7 also indicates that the change of the cosolvent-induced equilib-
rium shift (left-hand side of Equation 11.10) is predominantly determined by
