Chemistry 340B

Solutions to the First Examination

Fall 2003
3)
a)

C p =

(H ) H 1 H 2
=
T
T1 T2

Cp = (133.84-95.84)/(56.0-45.9) = 3.76 kcal/mol

b)
Cp arises because of the change in accessible non-polar surface area due to
protein unfolding. The ordered water around the larger non-polar surface area of the
denatured form requires extra heat to melt.
c)

Go(T) = Ho(Tm) [ 1 - T/Tm ] + CP( T - Tm) + CPTln(Tm/T)

G = 133.84(1-298/329) + 3.76(298-329) + 3.76298ln(329/298)
= 12.611 + (116.56) + 110.887
= 6938 cal/mol
= 6.94 kcal/mol

d)
G(GuHCl) = G(H2O) m[GuHCl]
At 3.0M GuHCl, the protein is 50% unfolded, G(GuHCl) = 0.
0 = 6.938 m (3)
m =2.31
G(GuHCl) = RTlnK
K=

0.9
0.1

0.002298ln9 =6.938 2.313[GuHCl]

[GuHCl] = 3.57 M

4.

GoGuHCl = GoH2O - m[GuHCl]

Equation 1: 0 = GoH2O - m [4]

Equation 2: -1.31 kcal/mol = GoH2O - m [4.8]
Solve these two equations for m and GoH2O
1 - 2 m 1.64
GoH2O = 6.56 kcal/mol
20%
For 20% denaturation, GGuHCl
= -RT ln

20
= 0.83 kcal/mol.
80

20%
[GuHCl]20% denaturation = ( GoH2O - G GuHCl
m)

= 3.5 M.
at [GuHCl] of 3.5 M, the protein is 20% denatured.
5) Guanidinium is proposed to denature proteins by binding directly to the polypeptide,
displacing water from hydration sites on the polypeptide. The guanidine can also bind to
the solvent, water, which is hydration, and their is an equilibrium for guanidine binding
to the protein or being hydrated in the bulk solution. The evidence of weak hydration is
consistent with the model that the guanidine does not interact strongly with water and,
hence, is preferably bound to the protein.
6) a. Cp results from the increase in the amount of water associated with non-polar
surface area when the protein unfolds. Hence, if there is a larger protein, the amount of
non-polar surface area exposed to the solvent upon unfolding of the protein will also be
larger.
b) None of the proteins will unfold at 0oC. The low temperature where Gou is zero
for each of the proteins is far below 273oK for these proteins.
c) The three domains unfold independently and, hence, the Tm will be the same even
though there are three domains linked in the protein. The values of Ho and Cp will be
expressed per mol of Sso3 and will, therefore, be three times the values determined for
the initial Sso7d domain.
Tm = 97.8 oC
Ho = 190.2 kcal/mol
Cp = 1.86 kcal/moloC

d) In the vant Hoff plot the data will all apply to the cooperative unit, which is the single
Sso7d domain. Hence, the values will be identical to those determined initially for this
domain
Tm = 97.8 oC
Ho = 63.4 kcal/mol
Cp = 0.62 kcal/moloC
e) The shorter loops in the thermophilic protein means that the size of the protein is less
(few amino acids). This will reduce the value of Ho and Cp since these apply per mol
of protein. The net result will be to increase the value of Tm, other things being equal.
Themophilic proteins have other changes, in addition to this, that increase the Tm.